MiR-30-regulated autophagy mediates angiotensin II-induced myocardial hypertrophy.

Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group). Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene), and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy

miR-34a modulates angiotensin II-induced myocardial hypertrophy by direct inhibition of ATG9A expression and autophagic activity.

Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3'-UTR, but not to the mutated 3'-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity

The percentage of autophagic vacuoles varies with <i>beclin-1</i> gene expression.

<p>The autophagic vacuoles was analyzed by calculating MDC-stained cardiomyocytes using flow cytometry. (<b>A, C</b> and <b>E</b>) Representative percentage of autophagic vacuoles measured in MDC-stained cardiomyocytes using flow cytometry. (<b>B</b>) Evaluation of the influence of 3-MA on autophagic vacuoles. Con: control group, Ang II: treated with 1 µmol/L Angiotensin II, and Ang II+3-MA: treated with 1 µmol/L Angiotensin II and 10 mmol/L 3-MA. *<i>P</i><0.05 compared with Con group; # <i>P</i><0.05 compared with Ang II group. (<b>D</b>) Evaluation of the influence of <i>beclin-1</i> specific siRNA on autophagic vacuoles. NC: treated with lentivirus containing negative control of <i>beclin-1</i>-specific siRNA, Ang II+ NC: treated with lentivirus containing negative control of <i>beclin-1</i>-specific siRNA and 1 µmol/L Angiotensin II, and Ang II+siRNA: treated with lentivirus containing <i>beclin-1</i>-specific siRNA and 1 µmol/L Angiotensin II. *<i>P</i><0.05 compared with NC group; # <i>P</i><0.05 compared with Ang II+NC group. (<b>F</b>) Evaluation of the influence of pRc/CMV2-beclin-1 vector on autophagic vacuoles. pRc/CMV2: transfected with pRc/CMV2 vector, and pRc/CMV2-beclin-1: transfected with pRc/CMV2-beclin-1. *<i>P</i><0.05 compared with pRc/CMV2 group. Data are presented as means ± SEM.</p

clinical characteristics of patients with and without LVH.

<p>DM = diabetes mellitus, ACS = acute coronary syndrome, Af = atrial fibrillation, SBP = systolic blood pressure, DBP = diastolic blood pressure, TC = total cholesterol, TG = total glyceride, HDL = high-density lipoprotein, LDL = low-density lipoprotein, Cr = creatinine, CK-MB = creatine kinase-MB, LV = left ventricular diameter.</p

Down-regulation of miR-30 leads to myocardial hypertrophy.

<p>(<b>A</b>) Relative expression of miR-30a in Ang II-stimulated cardiomyocytes (treated with 1 µmol/L Angiotensin II) and that in unstimulated cells by real-time RT-PCR relative to U6. *<i>P</i><0.05 compared with Con group. (<b>B</b>) Evaluation of the influence of miR-30a on mRNA level of <i>ANP</i> and <i>β-MHC</i> in hypertrophic cardiomyocytes. *<i>P</i><0.05 compared with Ang II+NC group. (<b>C</b>) Morphological changes were observed using confocal microscopy in cardiomyocytes stained with Alexa Fluor®555 Phalloidin and DAPI. Summarized data are shown in (<b>D</b> and <b>E</b>). (<b>D</b>) Evaluation of the influence of Ang II on relative cell area (the cardiac muscle fiber surface area ratio) in cardiomyocytes. *<i>P</i><0.05 compared with Con group. (<b>E</b>) Evaluation of the influence of miR-30a on relative cell area in hypertrophic cardiomyocytes. *<i>P</i><0.05 compared with Ang II+NC group.</p

circulating miR-30 expression in rats from the TAAC group.

<p>The circulating miR-30a in rats (miR-30a in Rattus norvegicus, rno-miR-30a) from TAAC group and Sham group was analyzed by real-time RT-PCR 4 weeks after the operation. cel-miR-39 was used an endogenous control. *<i>P</i><0.05 compared with Sham group.</p

MiR-30a regulates the percentage of autophagic vacuoles.

<p>The autophagic vacuoles was analyzed by calculating MDC-stained cardiomyocytes using flow cytometry. (<b>A</b>)Representative percentage of autophagic vacuoles measured in MDC-stained cardiomyocytes using flow cytometry. (<b>B</b>) Evaluation of the influence of miR-30a mimics on autophagic vacuoles. *<i>P</i><0.05 compared with Ang II +NC group. (<b>C</b>) Evaluation of the influence of miR-30a inhibitors on autophagic vacuoles. *<i>P</i><0.05 compared with NC group.</p

Expression of autophagy-related protein in cardiomyocytes varies with that of the <i>beclin-1</i> gene.

<p>The relative expression of autophagy-related protein in cardiomyocytes was analyzed by Western blotting. GAPDH was used as an internal control. Data are presented as means ± SEM. (<b>A</b>) Evaluation of the influence of 3-MA on the expression of LC3II/LC3I and beclin-1 proteins. Con: control group, Ang II: treated with 1 µmol/L Angiotensin II, and Ang II+3-MA: treated with 1 µmol/L Angiotensin II and 10 mmol/L 3-MA. *<i>P</i><0.05 compared with Con group; # <i>P</i><0.05 compared with Ang II group. (<b>B</b>) Evaluation of the influence of <i>beclin-1</i> specific siRNA on the expression of autophagy-related proteins. NC: treated with lentivirus containing negative control of <i>beclin-1</i>-specific siRNA, Ang II+ NC: treated with lentivirus containing negative control of <i>beclin-1</i>-specific siRNA and 1 µmol/L Angiotensin II, and Ang II+siRNA: treated with lentivirus containing <i>beclin-1</i>-specific siRNA and 1 µmol/L Angiotensin II. *<i>P</i><0.05 compared with NC group; # <i>P</i><0.05 compared with Ang II+NC group. (<b>C</b>) Evaluation of the influence of pRc/CMV2-beclin-1 vector on LC3II/LC3I and beclin-1 proteins. pRc/CMV2: transfected with pRc/CMV2 vector, and pRc/CMV2-beclin-1: transfected with pRc/CMV2-beclin-1. *<i>P</i><0.05 compared with pRc/CMV2 group.</p

Autophagy and miR-30 expression in rats after TAAC surgery.

<p>The relative expression of mRNA level, microRNA level, protein, and autophagic vacuoles in left ventricular tissues from Sham and TAAC rats was analyzed 4 weeks after the operation. (<b>A</b>) Relative mRNA expression level of <i>beclin-1</i> was analyzed by qRT-PCR. 18S was used as an internal control. (<b>B</b>) Relative expression of autophagy-related proteins, beclin-1 and LC3, was analyzed by Western blotting. GAPDH was used as an internal control. (<b>C</b>) Autophagic vacuoles (arrows indicated) were detected by transmission electron microscopy, at a magnification of ×13500. Scale bar: 1 µm. (<b>D</b>) Relative miR-30a, miR-30b and miR-30c expression which was presented as 2^−Δct*10⁻³ was analyzed by qRT-PCR. U6 was used as an internal control. Data are presented as means ± SEM. *<i>P</i><0.05 compared with Sham.</p

Relationship between the circulating miR-30 level and ventricular wall thickness.

<p>22 subjects were divided into two groups: LVH group (n = 11) and control group (n = 11). (<b>A</b>) Comparison of the circulating miR-30a (miR-30a in Homo sapiens, has-miR-30a) between patients with LVH and patients without LVH by real-time RT-PCR relative to cel-miR-39. *<i>P</i><0.05 compared with Con group. (<b>B</b>) Evaluation of the sensitivity and specificity of has-miR-30a on the diagnosis of LVH was performed by analyzing the ROC curve using MedCalc11.3 software. (<b>C</b>) The association between circulating has-miR-30a level and ventricular wall thickness was assessed by Pearson correlation analysis. <i>R</i>: Pearson correlation coefficient.</p