miR-15b over expression in HL-1 cells compromises mitochondrial membrane potential.

<p>(<b>A</b>,<b>B</b>) HL-1 cells were transfected (72 hours) with mimic negative control or miRNA-15b mimic. Untransfected cells were used as negative control and cells transfected with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive control (left panel –8 nM tetramethylrhodamine methyl ester (TMRM); center panel - 0.5 µl/ml plasma membrane potential indicator (PMPI); right panel - merged image). Bar graph shows significant decrease in TMRM with no change in PMPI. Solid bars represent mimic negative control while open bars represent miRNA-15b mimic. (<b>C</b>,<b>D</b>) Loss of mitochondrial membrane potential in HL-1 cells transfected with miRNA-15b mimic, as assessed by JC-1 flow cytometry 72 h post-transfection. Cells were transfected with a (i) mimic control, (ii) miRNA-15b mimic or (iii) treated with CCCP Arrows (K gate) indicate cells containing JC-1 aggregates resulting from intact mitochondria; M gate indicates cells with low or collapsed mitochondrial membrane potential. Ratio of polarized to depolarized cells calculated as a ratio between cells in K gate to M gate. Data indicate decrease in membrane potential upon up-regulation of miRNA-15b. (n = 3).</p

Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction

<div><p>Background</p><p>Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.</p><p>Results</p><p>Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.</p><p>Conclusions</p><p>Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.</p></div

Dicer deletion in the adult heart leads to oxidative stress.

<p>(<b>A</b>) Representative EPR imaging of nitroxide radical decay in (A) Dicer^+/+ and (B) Dicer^−/− hearts. (<b>B</b>) Time course of average % signal change in the region of interest (ROI). Logarithmic values of signal change (normalized to the initial signal at time = 0) in the ROIs are plotted with respect to time. Decay rate constants were obtained from the slope of linear decay after peak. Line with black circles represent Dicer^+/+ and line with grey circles represent Dicer^−/−. (<b>C</b>) Bar-graph showing the measured rate constants of nitroxide reduction in the tissues. (<b>D</b>) Thiobarbituric acid–reactive substances (TBARS), an indicator of lipid peroxidation was measured from Dicer^+/+ and Dicer^−/− hearts and was significantly higher in the later. (<b>E</b>) Total GSSG to GSH ratio in Dicer^+/+ mice heart compared to Dicer^−/− heart. (<b>F</b>) Lactate levels measured in Dicer^+/+ and Dicer^−/− mice hearts.</p

Suppression of induced miRNA-15b attenuates loss of Pim-1 and improves mitochondrial integrity.

<p>Immunohistochemical comparison of (<b>A</b>) Dicer (<b>B</b>) ANF, (<b>C</b>) Pim-1(<b>D</b>) ANT-1 (<b>E</b>) Comparison of cardiac histopathology sections stained with hematoxylin and eosin (H&E) (<b>F</b>) Bar graph showing quantification of IHC images. Image analysis software (Axiovision 4.3, Zeiss, Germany) was used to quantify fluorescence intensity (fluorescent pixels) and analyzed as a percent change in relative fluorescence unit (RFU). (n = 3) (<b>G</b>) Comparison between % of total mitochondria belonging to Class I/II or Class III/IV. (<b>H</b>) Representative TEM images of mitochondria from Dicer depleted heart tissue treated with anti-miR-15b. <i>Bar graphs represent equal SD on both sides of the mean.</i></p

Suppression of induced miRNA-15b prevents impairment of cardiac function.

<p>(<b>A</b>) Reduced expression of miRNA-15b upon anti-miRNA-15b delivery. (<b>B)</b> Representative M-mode images of Dicer^−/− and anti-miRNA-15b injected Dicer^−/− mice. (<b>C</b>) 11.7T MRI images of long axis at diastole (i & ii) and systole (iii & iv) (<b>D</b>) 11.7T images of short axis at diastole and systole of 1.0 mm slices stacked along columns from base to apex of the left ventricle as explained in Fig. 1. (<b>E</b>) (i) Fractional shortening, (ii) ejection fraction (EF) and (iii) LV mass. (<b>F</b>) (i) Fractional shortening, (ii) ejection fraction and (iii) LV mass in saline or anti-miR-15b injected Dicer+/+ (wild type).</p

Cardiac specific Dicer deletion leads to cardiac hypertrophy and impaired cardiac function.

<p>(<b>A</b>) Generation of Myh6-cre/Esr1-Dicer^fl/fl mice. (<b>B</b>) Western Blot showing reduced Dicer expression following tamoxifen injection. (<b>C</b>) 11.7T images of short axis at diastole and systole of 1.0 mm slices stacked along columns from base to apex of the left ventricle of both Dicer^+/+ and Dicer^−/− mice. Blue contour lines indicate LV volume. (<b>D</b>) Representative M-mode images of Dicer^+/+ and Dicer^−/− mice. The data indicates decreased contractility and increased left ventricular (LV) internal dimensions in Dicer^−/− mice. 11.7T MRI images of long axis at diastole (i & iii), systole (ii & iv) cardiac 11.7T MRI images in Dicer^+/+ and Dicer^−/− mice. The diastole of Dicer^−/− mice was enlarged due to insufficient contraction of the heart (shown by arrows). (<b>E</b>) Quantification of LV fractional shortening, LV myocardial mass, and LV ejection fraction (EF) by MRI and ECHO. Solid bars represent Dicer^+/+ and open bars represent Dicer^−/−. (<b>F</b>) (i) Ejection fraction, (ii) fractional shortening and (iii) LV mass in wild type corn oil or tamoxifen injected mice.</p

Dicer deletion in the adult heart leads to mitochondrial dysfunction.

<p>(<b>A</b>) TMRM assay in HL-1 cells transfected with si-control and si-Dicer. (<b>B</b>) TMRM assay on Dicer^+/+ and Dicer^−/− heart sections. Bar graph quantifying TMRM fluorescence demonstrates lower TMRM fluorescence in Dicer^−/− hearts (n = 3). (<b>C</b>) Representative TEM images of mitochondria from heart tissue. Arrows indicate mitochondria. Single mitochondria in white boxes have been zoomed into in the right panel. Labels <i>a)</i> indicate increased inter-cristae space as compared to the mitochondria in the Dicer^+/+ heart and <i>b)</i> points to loss of integrity of the cristae structure. (<b>D</b>) Representative diagram showing experiment for RCR measurements from isolated mitochondria. (<b>E</b>) Respiratory coupling ratio (RCR) was significantly lower in Dicer^−/− mice indicating mitochondrial dysfunction.</p

Pim-1 is a target of miRNA-15b and protein expression is reduced in Dicer^−/− mice.

<p>(<b>A</b>) Western blot analysis of Pim-1 protein from whole tissue homogenates from Dicer^+/+ and Dicer^−/− mice hearts. (<b>B</b>) Immunohistochemistry showing reduced Pim-1 expression in Dicer^−/− cardiac tissue. (<b>C</b>) Western blot analysis of Pim-1 protein from HL-1 cells transfected with si-control, si-Dicer and si-Dicer with miR-15b inhibitor. Quantification of Pim-1 expression was performed using densitometry. (<b>D</b>) Western blot analysis of Pim-1 protein from HL-1 cells transfected with scrambled control and miRNA-15b mimic, (<b>E</b>) Plasmid construct for pLuc-Pim1–3′UTR plasmid. (<b>F</b>) miRNA target reporter luciferase assay with Pim-1 3′UTR. Results were normalized with data obtained from an assay with <i>Renilla</i> luciferase. (<b>G</b>) Luciferase activity in HL-1 cells transfected with a Pim1-mutated 3′UTR and miR-15b mimic.</p

Cardiac specific <i>Dicer</i> deletion leads to early onset changes in some miRNAs including miRNA-15b.

<p>(<b>A</b>) Heat map generated for the differentially expressed miRNAs in Dicer^+/+ and Dicer^−/− mice. (<b>B</b>)Validation of selected miRNAs by real-time PCR. (<b>C</b>) miR-15b expression in laser captured cardiomyocytes from Dicer^+/+ and Dicer^−/− tissues.</p

WED impairs cell viability.

<p>CLSM micrographs of mature PAO1 biofilm stained with live-dead stain after treatment with placebo, WED or Ag control. The green fluorescence indicates live bacteria while the red indicates dead bacteria, n = 3.</p