Novel approaches for gene-specific interference via manipulating actions of microRNAs: examination on the pacemaker channel genes HCN2 and HCN4

Recent evidence has suggested microRNAs as viable therapeutic targets for a wide range of human disease. However, lack of gene-specificity of microRNA actions may hinder this application. Here we developed two new approaches, the gene-specific microRNA mimic and microRNA-masking antisense approaches, to explore the possibility of using microRNA's principle of actions in a gene-specific manner. We examined the value of these strategies as rational approaches to develop heart rate-reducing agents and "biological pacemakers" by manipulating the expression of the cardiac pacemaker channel genes HCN2 and HCN4. We showed that the gene-specific microRNA mimics, 22-nt RNAs designed to target the 3'untranslated regions (3'UTRs) of HCN2 and HCN4, respectively, were efficient in abrogating expression and function of HCN2 and HCN4. The gene-specific microRNA mimics repressed protein levels, accompanied by depressed f-channel conductance and the associated rhythmic activity, without affecting mRNA levels of HCN2 and HCN4. Meanwhile, we also designed the microRNA-masking antisense based on the miR-1 and miR-133 target sites in the 3'UTRs of HCN2 and HCN4 and found that these antisense oligodeoxynucleotides markedly enhanced HCN2/HCN4 expression and function, as reflected by increased protein levels of HCN2/HCN4 and If conductance, by removing the repression of HCN2/HCN4 expression induced by endogenous miR-1/miR-133. The experimental examination of these techniques and the resultant findings not only indicate feasibility of interfering miRNA action in a gene-specific fashion but also may provide a new research tool for studying function of miRNAs. The new approaches also have the potential of becoming alternative gene therapy strategies.     

MicroRNA-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiac myocytes

GLUT4 shows decreased levels in failing human adult hearts. We speculated that GLUT4 expression in cardiac muscle may be fine-tuned by microRNAs. Forced expression of miR-133 decreased GLUT4 expression and reduced insulin-mediated glucose uptake in cardiomyocytes. A computational miRNA target prediction algorithm showed that KLF15 is one of the targets of miR-133. It was confirmed that over-expression of miR-133 reduced the protein level of KLF15, which reduced the level of the downstream target GLUT4. Cardiac myocytes infected with lenti-decoy, in which the 3′UTR with tandem sequences complementary to miR-133 was linked to the luciferase reporter gene, had decreased miR-133 levels and increased levels of GLUT4. The expression levels of KLF15 and GLUT4 were decreased at the left ventricular hypertrophy and congestive heart failure stage in a rat model. The present results indicated that miR-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiomyocytes.     

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.     

IGF-1 deficiency resists cardiac hypertrophy and myocardial contractile dysfunction: role of microRNA-1 and microRNA-133a

This study was designed to examine the impact of insulin-like growth factor-1 (IGF-1) deficiency on abdominal aortic constriction (AAC)-induced cardiac geometric and functional changes with a focus on microRNA-1, 133a and 208, which are specially expressed in hearts and govern cardiac hypertrophy and stress-dependent cardiac growth. Liver-specific IGF-1-deficient (LID) and C57/BL6 mice were subject to AAC. Echocardiographic and cardiomyocyte function were assessed 4 wks later. Haematoxylin and eosin staining was used to monitor myocardial morphology. Western blot and real-time PCR were used to detect protein and miR expression, respectively. Neonatal rat cardiomyocytes (NRCMs) were transfected with miRs prior to IGF-1 exposure to initiate cell proliferation. Immunohistochemistry and [(3)H] Leucine incorporation were used to detect cell surface area and protein abundance. C57 mice subject to AAC displayed increased ventricular wall thickness, decreased left ventricular end diastolic and end systolic dimensions and elevated cardiomyocyte shortening capacity, all of which were attenuated in LID mice. In addition, IGF-1 deficiency mitigated AAC-induced increase in atrial natriuretic factor, GATA binding protein 4, glucose transporter 4 (GLUT4) and Akt phosphorylation. In contrast, neither AAC treatment nor IGF-1 deficiency affected glycogen synthase kinase 3b, mammalian target of rapamycin, the Glut-4 translocation mediator Akt substrate of 160 kD (AS160) and protein phosphatase. Levels of miR-1 and -133a (but not miR-208) were significantly attenuated by AAC in C57 but not LID mice. Transfection of miR-1 and -133a obliterated IGF-1-induced hypertrophic responses in NRCMs. Our data suggest that IGF-1 deficiency retards AAC-induced cardiac hypertrophic and contractile changes via alleviating down-regulation of miR-1 and miR-133a in response to left ventricular pressure overload.     

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.     

Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133

The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.     

Choline Protects Against Cardiac Hypertrophy Induced by Increased After-load

Background: Although inadequate intake of essential nutrient choline has been known to significantly increase cardiovascular risk, whether additional supplement of choline offering a protection against cardiac hypertrophy remain unstudied.Methods: The effects of choline supplements on pathological cardiac hypertrophic growth induced by transverse aorta constriction (TAC) for three weeks and cardiomyocyte hypertrophy in cultured cells induced by isoproterenol (ISO) 10 μM for 48 h stimulation were investigated. Western blot analysis and real-time PCR were used to determine the expression of ANP, BNP, β-MHC, miR-133a and Calcineurin.Results: Administration of 14 mg/kg choline to mice undergone TAC effectively attenuated the cardiac hypertrophic responses, as indicated by the reduced heart weight, left ventricular weight, ventricular thickness, and reduced expression of biomarker genes of cardiac hypertrophy. This anti-hypertrophic efficacy was reproduced in a cellular model of cardiomyocyte hypertrophy induced by isoproterenol in cultured neonatal cardiomyocytes. Our results further showed that choline rescued the aberrant downregulation of the muscle-specific microRNA miR-133a expression, a recently identified anti-hypertrophic factor, and restored the elevated calcineurin protein level, the key signaling molecule for the development of cardiac hypertrophy. These effects of choline were abolished by the M₃ mAChR-specific antagonist 4-DAMP.Conclusion: Our study unraveled for the first time the cardioprotection of choline against cardiac hypertrophy, with correction of expression of miR-133a and calcineurin as a possible mechanism. Our findings suggest that choline supplement may be considered for adjunct anti-hypertrophy therapy.     

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.     

Identification of miR-1293 potential target gene: TIMP-1

miRNAs play an important role in the pathogenesis of cardiac hypertrophy and dysfunction. However, little is known about how miR-30a regulates cardiomyocyte hypertrophy. In the study, Male C57BL/6 mice were subjected to thoracic aortic constriction, and hearts were harvested at 3 weeks. We assayed miR-30a expression level by real-time PCR and defined the molecular mechanisms of miR-30a-mediated cardiomyocyte hypertrophy. We found that myocardial expression of miR-30a was decreased in mouse models of hypertrophy and in H9c2 cells treated with phenylephrine. MiR-30a inhibition markedly increased mRNA expression of cardiac hypertrophy markers such as atrial natriuretic factor and brain natriuretic peptide in H9c2, and cell size was increased after miR-30a inhibitor treatment. Downregulated miR-30a activated autophagy by inhibiting beclin-1 expression in H9c2 cell. More important, autophagy inhibition suppressed miR-30a inhibitor-induced cardiomyocyte hypertrophy. Together, our data demonstrated that downregulated miR-30a aggravates pressure overload-induced cardiomyocyte hypertrophy by activating autophagy, thus offering a new target for the therapy of cardiomyocyte hypertrophy.     

Adiponectin Upregulates MiR-133a in Cardiac Hypertrophy through AMPK Activation and Reduced ERK1/2 Phosphorylation

Adiponectin and miR-133a are key regulators in cardiac hypertrophy. However, whether APN has a potential effect on miR-133a remains unclear. In this study, we aimed to investigate whether APN could regulate miR-133a expression in Angiotensin II (Ang II) induced cardiac hypertrophy in vivo and in vitro. Lentiviral-mediated adiponectin treatment attenuated cardiac hypertrophy induced by Ang II infusion in male wistar rats as determined by reduced cell surface area and mRNA levels of atrial natriuretic peptide (ANF) and brain natriuretic peptide (BNP), also the reduced left ventricular end-diastolic posterior wall thickness (LVPWd) and end-diastolic interventricular septal thickness (IVSd). Meanwhile, APN elevated miR-133a level which was downregulated by Ang II. To further investigate the underlying molecular mechanisms, we treated neonatal rat ventricular myocytes (NRVMs) with recombinant rat APN before Ang II stimulation. Pretreating cells with recombinant APN promoted AMP-activated protein kinase (AMPK) phosphorylation and inhibited ERK activation. By using the inhibitor of AMPK or a lentiviral vector expressing AMPK short hairpin RNA (shRNA) cancelled the positive effect of APN on miR-133a. The ERK inhibitor PD98059 reversed the downregulation of miR-133a induced by Ang II. These results indicated that the AMPK activation and ERK inhibition were responsible for the positive effect of APN on miR-133a. Furthermore, adiponectin receptor 1 (AdipoR1) mRNA expression was inhibited by Ang II stimulation. The positive effects of APN on AMPK activation and miR-133a, and the inhibitory effect on ERK phosphorylation were inhibited in NRVMs transfected with lentiviral AdipoR1shRNA. In addition, APN depressed the elevated expression of connective tissue growth factor (CTGF), a direct target of miR-133a, through the AMPK pathway. Taken together, our data indicated that APN reversed miR-133a levels through AMPK activation, reduced ERK1/2 phosphorylation in cardiomyocytes stimulated with Ang II, revealing a previously undemonstrated and important link between APN and miR-133a.     

MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.