Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure - PubMed (original) (raw)

. 2008 Feb 12;105(6):2111-6.

doi: 10.1073/pnas.0710228105. Epub 2008 Feb 6.

Elizabeth P Murchison, Ruhang Tang, Thomas E Callis, Mariko Tatsuguchi, Zhongliang Deng, Mauricio Rojas, Scott M Hammond, Michael D Schneider, Craig H Selzman, Gerhard Meissner, Cam Patterson, Gregory J Hannon, Da-Zhi Wang

Affiliations

Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure

Jian-Fu Chen et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Cardiovascular disease is the leading cause of human morbidity and mortality. Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy associated with heart failure. Here, we report that cardiac-specific knockout of Dicer, a gene encoding a RNase III endonuclease essential for microRNA (miRNA) processing, leads to rapidly progressive DCM, heart failure, and postnatal lethality. Dicer mutant mice show misexpression of cardiac contractile proteins and profound sarcomere disarray. Functional analyses indicate significantly reduced heart rates and decreased fractional shortening of Dicer mutant hearts. Consistent with the role of Dicer in animal hearts, Dicer expression was decreased in end-stage human DCM and failing hearts and, most importantly, a significant increase of Dicer expression was observed in those hearts after left ventricle assist devices were inserted to improve cardiac function. Together, our studies demonstrate essential roles for Dicer in cardiac contraction and indicate that miRNAs play critical roles in normal cardiac function and under pathological conditions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Generation of cardiac-specific Dicer knockout mice. (a) The gene targeting strategy for cardiac-specific deletion of Dicer. (b) PCR genotyping analysis of P0 mice. Wild-type loxP alleles and Cre products are indicated (Upper). Western blot analysis of Dicer protein from wild-type (+/+) and Dicer mutant (−/−) hearts. GAPDH serves as a loading control (Lower). (c) Genotyping results of P0 offspring from MHCCre/+; Dicerflox/+, mice and Dicerflox/flox mice intercrossing. (d) miRNA microarray analysis of miRNA expression in P0 wild-type (+/+), Dicer heterozygous (+/−), and homozygous (−/−) mutant hearts. Red denotes high expression and green denotes low expression. (e and f) miRNA Northern blot analysis to detect the expression of precursor (Pre-) and mature miR-1 and -133 in P0 or embryonic wild-type (+/+) and Dicer mutant (−/−) hearts. tRNAs were used as a loading control. (g) Kaplan–Meier survival curves for wild-type (Wt) and Dicer knockout (Ko) mice. All Dicer mutant mice die before P4.

Fig. 2.

Fig. 2.

Cardiac-specific Dicer deletion leads to DCM. (a) Gross morphology of P0 wild-type (+/+) and Dicer mutant (−/−) hearts. (b) H&E staining of sagittal sections of P0 wild-type (+/+) and Dicer mutant (−/−) hearts. (c) Higher-magnification images from H&E stained left ventricular myocardium of P0 wild-type (+/+) and Dicer mutant (−/−) hearts. (d) Immunohistology of P0 wild-type (+/+) and Dicer mutant (−/−) hearts for cTnT (green). DAPI stains nuclei. (e) Higher-magnification images from cTnT immunohistology-stained left ventricular myocardium of P0 wild-type (+/+) and Dicer mutant (−/−) hearts. (f) Confocal microscopic images of cultured neonatal cardiomyocytes from wild-type (+/+) and Dicer mutant (−/−) hearts stained for cardiac α-TM. (g) Electron microscopic analysis of ventricular myocardium of wild-type (+/+) and Dicer mutant (−/−) hearts. There is substantially less organized myofibril in Dicer mutant hearts. (h) Higher magnification of electron micrographies of wild-type (+/+) and Dicer mutant (−/−) myocardium to show disarrayed sarcomere in mutant hearts. (i) Quantitative measurement of sarcomere length in wild-type (+/+) and Dicer mutant (−/−) hearts. (j) TUNEL staining (green) detected increased apoptosis in the left atrium and ventricle of P2 Dicer mutant (−/−) heart. Antibody against cTnT was used to label cardiomyocytes (red). DAPI stains nuclei (blue). (k) Increased apoptosis in the left ventricular wall of P2 Dicer mutant (−/−) hearts as revealed by TUNEL staining. (l) Quantitative measurement of TUNEL positive cells in P2 wild-type (+/+) and Dicer mutant (−/−) hearts.

Fig. 3.

Fig. 3.

Impaired cardiac function in Dicer mutant hearts. (a) M-mode echocardiograms of P0 wild-type (+/+) and Dicer mutant (−/−) mice. (b) Diagram to show the areas of ventricles examined in confocal immunohistochemistry images: area 1 for c and f; area 2 for d and g; area 3 for e and h. (c–h) Confocal microscopic images for Cx40 protein expression (red) in the ventricles of P0 wild-type (+/+) and Dicer mutant (−/−) mice. DAPI stains nuclei. (i) Western blot analyses of connexin protein expression in P0 wild-type (+/+) and Dicer mutant (−/−) mice. β-Tubulin serves as a loading control. (j and k) Confocal microscopic images of Cx45 in P0 wild-type (+/+) and Dicer mutant (−/−) hearts. Laminin marks the cell surface and DAPI stains nuclei.

Fig. 4.

Fig. 4.

Expression of cardiac contractile proteins in Dicer mutant hearts. (a–d) Confocal microscopic images of P0 wild-type (+/+) and Dicer mutant (−/−) hearts stained with MHC in apexes (a and b) or left ventricle (c and d). Note patches of myocardium that lack expression of MHC in Dicer mutant (−/−) hearts. DAPI stains nuclei. (e and f) Confocal microscopic images of P0 wild-type (+/+) and Dicer mutant (−/−) hearts stained with antibody against α-TM. Note dramatic decrease in the expression of α-TM in Dicer mutant (−/−) hearts. DAPI stains nuclei. (g and h) Western blot analyses of indicated proteins using protein extracts from P0 wild-type (+/+) and Dicer mutant (−/−) hearts. β-tubulin serves as a loading control.

Fig. 5.

Fig. 5.

Dicer protein expression in patients with failure or nonfailure hearts. Western blot analyses for Dicer from total protein extracts of ventricles of human subjects with end-stage heart failure before (lanes 1–4) or after (lanes 5–8) the application of LVAD, or nonfailure heart samples (lanes 9–11). GAPDH serves as a loading control.

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