Attenuation of microRNA-22 derepressed PTEN to effectively protect rat cardiomyocytes from hypertrophy

Cardiac hypertrophy, which is characterized by the enlargement of cell size, reactivation of fetal genes, remains one of the most important triggers to heart failure. Increasing evidence shows that microRNA (miRNA) is extensively involved in the pathogenesis of cardiac hypertrophy. But the effects of miRNAs on cardiomyocyte hypertrophy have not been completely solved yet. Here, we showed that a collection of miRNAs was aberrantly expressed in hypertrophic cardiomyocytes induced by phenylephrine (PE) or angiotensin II (Ang II). Among them, miR-22 was the most strikingly up-regulated miRNA. To investigate the role of miR-22 in hypertrophy, both over-expression and knock-down assays were performed on cardiomyocytes. The results showed that up-regulation of miR-22 significantly increased the cell size and markedly influenced the expression of hypertrophic markers, including induction of nppa and reduction of myh6. In contrast, reduction of miR-22 level attenuated either PE- or Ang II-induced hypertrophic reaction. Furthermore, several genes, including PTEN, were identified as potential targets of miR-22 by bioinformatic algorithms. Using luciferase analysis, miR-22 could significantly suppress the luciferase activity of reporter fused with 3' untranslated region of PTEN mRNA. Furthermore, up-regulation of miR-22 could suppress the protein level of PTEN and reduction of miR-22 level markedly increased the protein level of PTEN in cardiomyocytes by Western blot analysis, suggesting that the contribution of miR-22 to cardiomyocyte hypertrophy may be partially through targeting PTEN. Taken together, miRNAs were dynamically regulated in cardiomyocyte hypertrophy and attenuation of miR-22 in rat cardiomyocytes efficiently protected from hypertrophic effects through derepressing PTEN.     

Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.     

MiR-146b protect against sepsis induced mice myocardial injury through inhibition of Notch1

Myocardial dysfunction is a major cause of death in sepsis. MicroRNA-146b (miR-146b) has been reported to be related to myocardial disease. However, the role of miR-146b in sepsis as well as myocardial injury is still unclear. Septic cardiac dysfunction in mice was induced by cecal ligation and puncture (CLP) and miR-146b was found increased significantly in the myocardium tissue of CLP mice. It was found that up-regulation of miR-146b by agomiR injection suppressed expression of IL-1β in mice as well as myocardium apoptosis in CLP mice. However, suppression of miR-146b by antagomiR injection had inverse effects. Notch1 was identified as a target gene of miR-146b by bioinformatics analysis. And it was verified that in cardiomyocytes, the decrease of miR146b led to increase of both the mRNA and protein level of Notch1 and vice versa. In septic mice serum stimulated cardiomyocytes, up-regulation of miR-146b decreased the level of Notch1 and Hes1. The knockout of Notch1 in transgenic mice showed that the deficiency of Notch1 improved myocardial injury induced by CLP operation. The apoptosis of cardiomyocytes was relieved and the expression of IL-1β was decreased. In conclusion, miR-146b targets to Notch1 and protected cardiomyocytes against inflammation and apoptosis.     

ICS II protects against cardiac hypertrophy by regulating metabolic remodelling,not by inhibiting autophagy

Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization. Therefore, the regulation of ketone body uptake and metabolism may have beneficial effects on heart injuries that induce cardiac remodelling. In this study, we investigated whether icariside II (ICS II) protects against cardiac hypertrophy in mice and cardiomyocytes. To create cardiac hypertrophy animal and cell models, mice were subjected to transverse aortic constriction (TAC), and embryonic rat cardiomyocytes (H9C2) were stimulated with angiotensin II, a neurohumoral stressor. Both the in vivo and in vitro results suggest that ICS II treatment ameliorated pressure overload–induced cardiac hypertrophy and preserved heart function. In addition, apoptosis and oxidative stress were reduced in the presence of ICS II. Moreover, ICS II inhibited excess autophagy in TAC-induced hearts and angiotensin II–stimulated cardiomyocytes. Mechanistically, we found that ICS II administration regulated SIRT3 expression in cardiac remodelling. SIRT3 activation increased ketone body transportation and utilization. Collectively, our data show that ICS II attenuated cardiac hypertrophy by modulating ketone body and fatty acid metabolism, and that this was likely due to the activation of the SIRT3-AMPK pathway. ICS II treatment may provide a new therapeutic strategy for improving myocardial metabolism in cardiac hypertrophy and heart failure.     

HMGA1 is a new target of miR-195 involving isoprenaline-induced cardiomyocyte hypertrophy

Emerging data have shown that microRNAs (miRNAs) have important functions in the processes of cardiac hypertrophy and heart failure that occur during the postnatal period. Cardiac overexpression of miR-195 results in pathological cardiac growth and heart failure in transgenic mice. In the present study, we analyzed the roles of miR-195 in cardiomyocyte hypertrophy and found that miR-195 was greatly upregulated during isoprenaline-induced cardiomyocyte hypertrophy. By using mRNA microarray and molecular approach, we identified a novel putative target of miR-195 called high-mobility group A1 (HMGA1). Total mRNA microarray showed that HMGA1 was downregulated in primary cardiomyocytes that overexpressed miR-195. Using luciferase activity assay, we demonstrated that miR-195 interacts with the 3′-untranslated region of HMGA1 mRNA. Moreover, we showed that miR-195 in primary cardiomyocytes downregulates the expression of HMGA1 at the protein level. Taken together, our data demonstrated that miR-195 can negatively regulate a new target, HMGA1, which is involved in cardiomyocyte hypertrophy.     

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.     

microRNA-665 silencing improves cardiac function in rats with heart failure through activation of the cAMP signaling pathway

Heart failure (HF) is a disease with high mortality and morbidity rate. Previous studies have shown that microRNAs (miRNAs) may be implicated in the pathogenesis of HF, potentially being able to improve the cardiac function in an HF rat model. The present study was designed to define the role of miR-665 in the cardiac function of the HF rats. Following the establishm;ent of the rat models of HF, the functional role miR-665 in HF was determined using an ectopic expression and knockdown experiments. The cardiac function was evaluated with the determination of ventricular mass index and hemodynamic parameters. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was performed, with the apoptosis of cardiac cells detected in the process. The expression of miR-665, glucagon-like peptide 1 receptor (GLP1R), cyclic adenosine monophosphate (cAMP) signaling pathway-related, and apoptosis-related genes was examined. Enzyme-linked immunosorbent assay was conducted to determine the levels of inflammation-related genes. Initially, the upregulation of miR-665, downregulation of GLP1R, and inactivation of cAMP signaling pathway were observed in HF rats. GLP1R was a target of miR-665. Forced expression of miR-665 promoted cell apoptosis and inhibited GLP1R and the cAMP signaling pathway. In addition, miR-665 overexpression has been known to impair cardiac function, promote inflammatory response while elevating malondialdehyde and superoxide dismutase levels, and decreasing mitochondrial respiratory chain enzyme activities. Furthermore, we also observed that the effects of miR-665 inhibition had been reversed when the cAMP signaling pathway was also inhibited. This study demonstrates that miR-665 inhibition can stabilize the cardiac function of HF rats via the cAMP signaling pathway via upregulation of the GLP1R.     

Circ-Zfp644 acts as a pro-hypertrophic mediator in an Ang-II induced in vitro myocardial hypertrophy model

Cardiac hypertrophy (CH) is a common risk factor for heart failure and even sudden cardiac death. Molecules have emerged as vital regulators in cardiac disorders. LIM domain kinase 1 (Limk1) is reported as a pro-fibrotic mediator in patients with permanent atrial fibrillation and it has also been suggested to aggravate cardiac dysfunction in rats with chronic heart failure. The present study observed that Limk1 was significantly upregulated in the in vitro model of CH induced by angiotensin II (Ang-II). Interestingly, silencing Limk1 led to inhibition of the hypertrophic phenotypes in Ang-II-treated cardiomyocytes. Next, through a series of mechanistic assays including RIP assay, RNA pull-down assay, and luciferase reporter assay, miR-93-5p was verified to target Limk1. Furthermore, circ-Zfp644 was validated as the sponge of miR-93-5p. Circ-Zfp644 functioned as a ceRNA to upregulate Limk1 expression via sequestering miR-93-5p in Ang-II-treated cardiomyocytes. Finally, a range of rescue assays revealed that circ-Zfp644 stimulated hypertrophic effects in Ang-II-treated cardiomyocytes via upregulating Limk1 while miR-93-5p exerted the opposite effects via its inhibition on Limk1. On the whole, the present study revealed that circ-Zfp644 aggravated CH through modulating the miR-93-5p/Limk1 axis. The findings observed on the in vitro model of CH provided new potential for developing CH treatment.     

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.     

Manipulation of Cardiac Phosphatidylinositol 3-Kinase (PI3K)/Akt Signaling by Apoptosis Regulator through Modulating IAP Expression (ARIA) Regulates Cardiomyocyte Death during Doxorubicin-induced Cardiomyopathy

PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.     

Diacylglycerol kinase-epsilon restores cardiac dysfunction under chronic pressure overload: a new specific regulator of Galpha(q) signaling cascade

Galpha(q) protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGKepsilon acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGKepsilon prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGKepsilon (DGKepsilon-TG) using an alpha-myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGKepsilon-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGKepsilon-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGKepsilon-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGKepsilon-TG mice, and the survival rate after TAC was higher in DGKepsilon-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGKepsilon-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGKepsilon-TG mice. In conclusion, these results demonstrate the first evidence that DGKepsilon restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGKepsilon may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.     

Cardiomyocyte Aldose Reductase Causes Heart Failure and Impairs Recovery from Ischemia

Aldose reductase (AR), an enzyme mediating the first step in the polyol pathway of glucose metabolism, is associated with complications of diabetes mellitus and increased cardiac ischemic injury. We investigated whether deleterious effects of AR are due to its actions specifically in cardiomyocytes. We created mice with cardiac specific expression of human AR (hAR) using the α–myosin heavy chain (MHC) promoter and studied these animals during aging and with reduced fatty acid (FA) oxidation. hAR transgenic expression did not alter cardiac function or glucose and FA oxidation gene expression in young mice. However, cardiac overexpression of hAR caused cardiac dysfunction in older mice. We then assessed whether hAR altered heart function during ischemia reperfusion. hAR transgenic mice had greater infarct area and reduced functional recovery than non-transgenic littermates. When the hAR transgene was crossed onto the PPAR alpha knockout background, another example of greater heart glucose oxidation, hAR expressing mice had increased heart fructose content, cardiac fibrosis, ROS, and apoptosis. In conclusion, overexpression of hAR in cardiomyocytes leads to cardiac dysfunction with aging and in the setting of reduced FA and increased glucose metabolism. These results suggest that pharmacological inhibition of AR will be beneficial during ischemia and in some forms of heart failure.     

LAZ3 protects cardiac remodeling in diabetic cardiomyopathy via regulating miR-21/PPARa signaling

Diabetes contributes to cardiovascular complications and the pathogenesis of cardiac remodeling that can lead to heart failure. We aimed to evaluate the functional role of LAZ3 in diabetic cardiomyopathy (DCM). Streptozotocin (STZ) was used to induce a diabetic mouse model. Three months after induction, the mice were subjected to retro-orbital venous plexus injection of adeno-associated virus 9 (AAV9) that overexpressed LAZ3. Six weeks after the infection, mouse hearts were removed to assess the degree of cardiac remodeling. LAZ3 was down-regulated in the diabetic mouse hearts and high glucose stimulated cardiomyocytes. Knock-down of LAZ3 in cardiomyocytes with LAZ3 siRNA reduced cell viability, increased the inflammatory response and induced oxidative stress and cell apoptosis. Overexpression of LAZ3 by infection with adeno-associated virus (AAV9)-LAZ3 protected against an inflammatory response, oxidative stress and cell apoptosis in both a high glucose stimulated in vitro study and diabetic mouse hearts. We found that LAZ3 increased the activation of PPARa, which increased PGC-1a activation and subsequently augmented NRF2 expression and nuclear translocation. This outcome was confirmed by NRF2 siRNA and a PPARa activator, since NRF2 siRNA abrogated the protective effects of LAZ3 overexpression, while the PPARa activator reversed the deteriorating phenotype of LAZ3 knock-down in both the in vitro and vivo study. Furthermore, LAZ3 decreased miR-21 expression, which resulted in PPARa activation, NRF2 expression and nuclear translocation. In conclusion, LAZ3 protects against cardiac remodeling in DCM by decreasing miR-21, thus regulating PPARa/NRF2 signaling.     

miR-885 mediated cardioprotection against hypoxia/reoxygenation-induced apoptosis in human cardiomyocytes via inhibition of PTEN and BCL2L11 and modulation of AKT/mTOR signaling

Ischemia/reperfusion (I/R) injury could cause the enhanced cell apoptosis of cardiomyocytes, which is one of key contributors for the development of ischemic heart disease. Recent studies emphasized the role of microRNAs (miRNAs) in regulating cardiomyocyte apoptosis. The study planned to elucidate the molecular actions of miR-885 on mediating human cardiomyocytes (HCMs) apoptosis induced by hypoxia/reoxygenation (H/R) and to explore the potential molecular mechanisms. The present data revealed that H/R stimulation inhibited HCM viability and potentiated HCM apoptosis, and more importantly, the expression of miR-885 in HCMs was markedly repressed after H/R stimulation. Further experimental examinations demonstrated that overexpression of miR-885 attenuated H/R-induced increased in HCM apoptotic rates, while miR-885 knockdown impaired HCM viability and increased HCM apoptotic rates. Moreover, the mechanistic studies showed that miR-885 inversely regulated the expression of phosphatase and tensin homolog (PTEN) and BCL2 like 11 (BCL2L11) in HCMs, and enforced expression of PTEN and BCL2L11 partially antagonized the protective actions of miR-885 overexpression on H/R-induced HCM injury. Moreover, H/R suppressed AKT/mTOR signaling, which was attenuated by miR-885 overexpression in HCMs. In conclusion, the present study for the first time showed the downregulation of miR-885 induced by H/R in HCMs, and provided the evidence that miR-885 attenuated H/R-induced cell apoptosis via inhibiting PTEN and BLC2L11 and modulation of AKT/mTOR signaling in HCMs.     

Cardiac-specific CGI-58 deficiency activates the ER stress pathway to promote heart failure in mice

Excess myocardial triacylglycerol accumulation (i.e., cardiac steatosis) impairs heart function, suggesting that enzymes promoting triacylglycerol metabolism exert essential regulatory effects on heart function. Comparative gene identification 58 (CGI-58) is a key enzyme that promotes the hydrolysis of triglycerides by activating adipose triglyceride lipase and plays a protective role in maintaining heart function. In this study, the effects of CGI-58 on heart function and the underlying mechanism were investigated using cardiac-specific CGI58-knockout mice (CGI-58^cko mice). Echocardiography and pathological staining were performed to detect changes in the structure and function of the heart. Proteomic profiling, immunofluorescent staining, western blotting, and real-time PCR were used to evaluate molecular changes. In CGI-58^cko mice, we detected cardiac hypertrophic remodeling and heart failure associated with excessive cardiac lipid accumulation, ROS production, and decreased expression of regulators of fatty acid metabolism. These changes were markedly attenuated in CGI-58^cko mice injected with rAAV9-CGI58. A quantitative proteomics analysis revealed significant increases in the expression of ER stress-related proteins and decreases in proteins related to fatty acid and amino acid metabolism in the hearts of CGI-58^cko mice. Furthermore, the inhibition of ER stress by the inhibitor 4-PBA improved mitochondrial dysfunction, reduced oxidative stress, and reversed cardiac remodeling and dysfunction in cultured cardiomyocytes or in CGI-58^cko mice. Our results suggested that CGI-58 is essential for the maintenance of heart function by reducing lipid accumulation and ER stress in cardiomyocytes, providing a new therapeutic target for cardiac steatosis and dysfunction.Subject terms: Heart failure, Heart failure