Die Rolle von kardialen MicroRNAs in der Pathogenese der dilatativen Kardiomyopathie

Zusammenfassend konnten in dieser Arbeit zwei microRNAs identifiziert werden, die signifikant im Myokard eines Mausmodells für die DCM dysreguliert sind. Es konnten Techniken zur Überexpression bzw. „knockdown“ dieser microRNAs entwickelt und erfolgreich in primären Kardiomyozyten angewendet werden. Die phänotypische und molekulare Analyse dieser Zellen erbrachte Hinweise, dass insbesondere die miR-301a eine wesentliche Rolle in der Pathogenese der DCM spielen könnte, da sie dem Tiermodell sehr ähnlich eine Dissoziation typischer molekularer Marker der Hypertrophie und Zellgröße verursachte

MicroRNA miR-301a is a novel cardiac regulator of Cofilin-2

<div><p>Calsarcin-1 deficient mice develop dilated cardiomyopathy (DCM) phenotype in pure C57BL/6 genetic background (Cs1-ko) despite severe contractile dysfunction and robust activation of fetal gene program. Here we performed a microRNA microarray to identify the molecular causes of this cardiac phenotype that revealed the dysregulation of several microRNAs including miR-301a, which was highly downregulated in Cs1-ko mice compared to the wild-type littermates. Cofilin-2 (Cfl2) was identified as one of the potential targets of miR-301a using prediction databases, which we validated by luciferase assay and mutation of predicted binding sites. Furthermore, expression of miR-301a contrastingly regulated Cfl2 expression levels in neonatal rat ventricular cardiomyocytes (NRVCM). Along these lines, Cfl2 was significantly upregulated in Cs1-ko mice, indicating the physiological association between miR-301a and Cfl2 <i>in vivo</i>. Mechanistically, we found that Cfl2 activated serum response factor response element (SRF-RE) driven luciferase activity in neonatal rat cardiomyocytes and in C2C12 cells. Similarly, knockdown of miR301a activated, whereas, its overexpression inhibited the SRF-RE driven luciferase activity, further strengthening physiological interaction between miR-301a and Cfl2. Interestingly, the expression of SRF and its target genes was strikingly increased in Cs1-ko suggesting a possible <i>in vivo</i> correlation between expression levels of Cfl2/miR-301a and SRF activation, which needs to be independently validated. In summary, our data demonstrates that miR-301a regulates Cofilin-2 <i>in vitro</i> in NRVCM, and <i>in vivo</i> in Cs1-ko mice. Our findings provide an additional and important layer of Cfl2 regulation, which we believe has an extended role in cardiac signal transduction and dilated cardiomyopathy presumably due to the reported involvement of Cfl2 in these mechanisms.</p></div

Model figure of cardiac role of Cfl2 and miR-301a.

<p>Our data from the present study illustrated involvement of Cfl2 in the activation of SRF signaling via RhoA. Both RhoA and Cfl2 plays important role in actin treadmilling. Increased levels of F-actin in turn activates SRF signaling. RhoA also activates SRF signaling through other effector molecules like myocardin-related transcription factors, etc.. On the other hand, miR-301a targets Cfl2 in cardiomyocytes, thereby inhibiting the activation of SRF signaling.</p

miR-301a and Cfl2 oppositely regulates Rho-mediated SRF signaling.

<p>Cfl2 or miR-301a were either overexpressed or knocked-down using respective vectors, mimic, or inhibitor transfection in C2C12 cells together with a luciferase construct carrying SRF-RE driven firefly luciferase. Overexpression of Cfl2 additively increased the luciferase activation by either constitutively RhoA (<b>A</b>), whereas, its siRNA led to inhibition of luciferase activation, basal, as well as in presence of RhoA (<b>B</b>). Knockdown of miR-301a also increased the luciferase activation (<b>C</b>), and the overexpression of miR-301a mimic significantly blunted the luciferase activity (<b>D</b>). For SRF-gene reporter assays in NRVCM, Cfl2 was overexpressed using adenovirus encoding rat Cfl2, whereas, its knockdown was achieved using adenovirus encoding synthetic microRNA specifically targeting Cfl2. Expression of miR-301a was modulated in NRVCM same as in C2C12 cells. Like in C2C12 cells, Overexpression of Cfl2 in NRVCM also exhibited positive effect on the activation of luciferase activity (<b>E</b>); knockdown of Cfl2 on the other hand significantly inhibited the activation of SRF-RE driven luciferase activity (<b>F</b>). Consistently, altered expression of miR-301a by treating NRVCM with miR-301a inhibitor (<b>G</b>) or mimic (<b>H</b>) oppositely affected the luciferase activation. Increased expression of Cfl2 in NRVCM did not alter cell surface area (<b>I</b>). Knockdown of miR-301a also led to no effect on cell size (<b>J</b>). Similarly, siRNA mediated knockdown of Cfl2 (<b>K</b>) or overexpression of miR-301a too did not alter cell surface area (<b>L</b>). N>500 for cell size measurements, and N = 6 luciferase assays. All experiments have been repeated at least twice. The statistical analysis was carried out using two-tailed student’s <i>t-test</i>. *: p<0.05, ‡: p<0.001, n.s.: non-significant.</p

Expression of miR-301a and Cfl2 is higher in cardiomyocytes compared to fibroblasts.

<p>Protein and RNA was extracted from isolated neonatal rat cardiomyocytes and fibroblasts for comparative analysis of miR-301a and Cfl2 expression status. (<b>A</b>) Expression levels of Cfl2 is found to be higher in cardiomyocytes compared to the fibroblasts shown in an immunoblot (original uncropped blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183901#pone.0183901.s002" target="_blank">S2B Fig</a>, respective densitometry is shown as a bar graph in <b>B</b>). (<b>C</b>) Expression of miR-301a was also higher in cardiomyocytes than fibroblasts as determined by quantitative real-time PCR. (<b>D</b>) α-actinin and vimentin levels were determined in the same samples as markers of cardiomyocytes and fibroblasts, respectively, to assess the purity of isolated cells (original uncropped blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183901#pone.0183901.s002" target="_blank">S2C Fig</a>, respective densitometry is shown as a bar graph in <b>E, F</b>). N = 4 each. The statistical analysis was carried out using two-tailed student’s <i>t-test</i>. †: p<0.01, ‡: p<0.001.</p

Cofilin-2 is a target of miR-301a.

<p>(<b>A</b>) We selected a subset of the targets identified through online microRNA target database search, including Cofilin-2 (Cfl2), Activin A Receptor Type 1 (ACVR1), Quaking (Qk), and Chloride Voltage-Gated Channel 3 (CLCN3). Putative 3’UTR binding sites from these targets was evaluated using pmirGLO Dual-Luciferase miRNA Target Expression Vector and assay system and found that only Cfl2 was a possible target (N = 6). (<b>B</b>) Cfl2 3’UTR has 4 putative binding sites (detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183901#pone.0183901.s001" target="_blank">S1A–S1D Fig</a>), which we mutated using site directed mutagenesis to confirm which of these binding sites act as targets for miR-301a binding. Mutations in binding sites 370 and 1030 resulted in loss of luciferase activation, clearly suggesting that these two sites act as binding sites for miR-301a (N = 4). (<b>C</b>) Immunoblot showing that the overexpression of miR-301a mimic downregulated (N = 3 each, original uncropped blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183901#pone.0183901.s001" target="_blank">S1B & S1C Fig</a>, respective densitometry is shown as a bar graph in <b>D</b>), whereas, overexpression of miR-301a inhibitor upregulated (respective densitometry is shown as a bar graph in <b>E</b>) the protein levels of Cfl2. (<b>F</b>) Cfl2 was found upregulated in Cs1-ko mice as depicted in an immunoblot at protein (N = 3 (WT), 4 (Cs1-ko), original uncropped blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183901#pone.0183901.s001" target="_blank">S1D Fig</a>, respective densitometry is shown as a bar graph in <b>G</b>), and at transcript level determined by immunoblotting and quantitative real-time PCR (<b>H</b>), respectively. All experiments are repeated at least two times and the statistical analysis was carried out using two-tailed student’s <i>t-test</i>. *: p<0.05, †: p<0.01, n.s.: non-significant.</p

Calsarcin-1 deficiency upregulates SRF in mouse heart.

<p>(<b>A</b>) SRF and RhoA are upregulated in Cs1-ko mice as depicted in an immunoblot (N = 3 (WT), 4 (Cs1-ko), original uncropped blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183901#pone.0183901.s003" target="_blank">S3 Fig</a>, respective densitometries are shown as bar graphs in <b>B</b> and <b>C</b>). Fetal gene program was strikingly activated in Cs1-ko mice as detected by increased expression of <i>nppa</i> (<b>D</b>), <i>nppb</i> (<b>E</b>), and <i>myh7</i> (<b>F</b>). Similarly cardiac muscle α-actin (<i>actc1</i>) was highly upregulated in Cs1-ko mice. N = 5 (WT) and 6 (Cs1-ko). The statistical analysis was carried out using two-tailed student’s <i>t-test</i>. *: p<0.05, †: p<0.01, ‡: p<0.001.</p

Phenotypic characterization of Calsarcin-1 deficient mice in C57BL/6 background.

<p>13 weeks old mice underwent echocardiography to assess cardiac function. Reduced fractional shortening (<b>A</b>), intraventricular diameter (IVD, <b>B</b>), and increased left ventricular end diastolic diameter (LVEDD, <b>C</b>) indicates contractile dysfunction and cardiac dilatation of Calsarcin-1 deficient mice (Cs1-ko) compared to wild-type (WT) littermates. Ratios of heart weight to body weight (<b>D</b>), and heart weight to tibia length (<b>E</b>) were unchanged between both genotypes suggesting no cardiac hypertrophy (N = 7 (WT), and 10 (Cs1-ko) for A-E). In line with unchanged heart weights, there was no difference in cardiomyocyte cell size in Cs1-ko or WT mice as determined by lectin staining (<b>F</b>), and measurement of cell surface area (<b>G</b>) (N >150 for G). (<b>H</b>) Bar graph indicating no difference in the fibrotic lesions in Cs1-ko mice compared to wild-type littermates (N = 3 each). Similarly, there was no difference in the expression levels of collagen I (<b>I</b>) and III (<b>J</b>), determined by quantitative real-time PCR. The statistical analysis was carried out using two-tailed student’s <i>t-test</i>. *: p<0.05, †: p<0.01, ‡: p<0.001, n.s.: non-significant.</p

MicroRNA miR-301a is downregulated in Cs1-ko mice.

<p>Microarray analyses were performed on Illumina Mouse Sentrix-6 BeadChip arrays (Illumina, Inc.) using total RNA isolated from Calsarcin knockout (Cs1-ko) and wild-type (WT) mice. Microarray scanning was done using a Beadstation array scanner and analyzed by normalization of the signals using the quantile normalization algorithm without background subtraction. Differentially regulated microRNAs were defined by calculating the standard deviation differences of a given probe in Cs1-ko and WT genotypes. (<b>A</b>) Bar graph presenting few selected dysregulated microRNAs in Cs1-ko mice compared to WT mice. MiR-301a was identified the most downregulated microRNA, whereas, miR-298 was highly upregulated (N = 4 each), which was confirmed in independent cohort by quantitative real-time PCR for miR-301a (<b>B</b>), and miR-298 (<b>C</b>) (N = 5 (WT), and 6 (Cs1-ko)). Statistical analysis was carried out using two-tailed student’s <i>t-test</i>. *: p<0.05, †: p<0.01.</p