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Cardiomyocyte-specific miRNA-30c over-expression causes dilated cardiomyopathy.

Wijnen WJ, van der Made I, van den Oever S, Hiller M, de Boer BA, Picavet DI, Chatzispyrou IA, Houtkooper RH, Tijsen AJ, Hagoort J, van Veen H, Everts V, Ruijter JM, Pinto YM, Creemers EE - PLoS ONE (2014)

Bottom Line: We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy.This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level.We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function.

View Article: PubMed Central - PubMed

Affiliation: Heart Failure Research Center, Academic Medical Center, Amsterdam, The Netherlands; Interuniversitair Cardiologisch Instituut Nederland (ICIN-NHI), Utrecht, The Netherlands.

ABSTRACT
MicroRNAs (miRNAs) regulate many aspects of cellular function and their deregulation has been implicated in heart disease. MiRNA-30c is differentially expressed in the heart during the progression towards heart failure and in vitro studies hint to its importance in cellular physiology. As little is known about the in vivo function of miRNA-30c in the heart, we generated transgenic mice that specifically overexpress miRNA-30c in cardiomyocytes. We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy. Gene expression analysis of the miRNA-30c transgenic hearts before onset of the phenotype indicated disturbed mitochondrial function. This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level. Taken together these data indicate impaired mitochondrial function due to OXPHOS protein depletion as a potential cause for the observed dilated cardiomyopathic phenotype in miRNA-30c transgenic mice. We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function.

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MiRNA-30c expression in the heart and the generation of αMHC-miRNA-30c transgenic mice (miRNA-30c TG).(a) MiRNA-30c in situ hybridization on adult wildtype hearts shows expression in the nuclei of both cardiomyocytes (black arrowheads) and interstitial cells (grey arrowheads). The cytoplasm is also miRNA-30c positive. (b) Schematic overview of the miRNA-30c overexpression construct used for the generation of transgenic mice. (c) Northern blot for miRNA-30c in wildtype and transgenic littermates in line B at 8 weeks of age. U6 was used as a loading control and shows similar loading. (d) Quantification of miRNA-30c overexpression by qPCR in both transgenic lines at 4 weeks of age (N≥6). (e) MiRNA-30c expression in left en right ventricular tissue of line B at 4 weeks of age (N = 6). Error bars represent s.e.m. and * denotes a p-value ≤0.05.
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pone-0096290-g001: MiRNA-30c expression in the heart and the generation of αMHC-miRNA-30c transgenic mice (miRNA-30c TG).(a) MiRNA-30c in situ hybridization on adult wildtype hearts shows expression in the nuclei of both cardiomyocytes (black arrowheads) and interstitial cells (grey arrowheads). The cytoplasm is also miRNA-30c positive. (b) Schematic overview of the miRNA-30c overexpression construct used for the generation of transgenic mice. (c) Northern blot for miRNA-30c in wildtype and transgenic littermates in line B at 8 weeks of age. U6 was used as a loading control and shows similar loading. (d) Quantification of miRNA-30c overexpression by qPCR in both transgenic lines at 4 weeks of age (N≥6). (e) MiRNA-30c expression in left en right ventricular tissue of line B at 4 weeks of age (N = 6). Error bars represent s.e.m. and * denotes a p-value ≤0.05.

Mentions: To examine the cell types expressing miRNA-30c in the heart in vivo, we performed in situ hybridization on hearts of wildtype mice using a Fam-labeled LNA probe against miRNA-30c (Figure 1a). This staining revealed expression of miRNA-30c in the nuclei of both cardiomyocytes (black arrowheads) and non-cardiomyocytes (grey arrowheads). Hybridization with the control probe displayed no signal. In addition, we found a diffuse pattern of miRNA-30c expression in the cytoplasm of cardiomyocytes which was not observed in sections treated with the control probe. The nuclear staining likely identifies pre-miRNA-30c while the cytoplasmic staining detects mature miRNA-30c. The expression of miRNA-30c in both cardiomyocytes and non-cardiomyocytes confirms previously reported in vitro studies [12], [13], which show expression in cultured neonatal myocytes and fibroblasts of the rat heart.


Cardiomyocyte-specific miRNA-30c over-expression causes dilated cardiomyopathy.

Wijnen WJ, van der Made I, van den Oever S, Hiller M, de Boer BA, Picavet DI, Chatzispyrou IA, Houtkooper RH, Tijsen AJ, Hagoort J, van Veen H, Everts V, Ruijter JM, Pinto YM, Creemers EE - PLoS ONE (2014)

MiRNA-30c expression in the heart and the generation of αMHC-miRNA-30c transgenic mice (miRNA-30c TG).(a) MiRNA-30c in situ hybridization on adult wildtype hearts shows expression in the nuclei of both cardiomyocytes (black arrowheads) and interstitial cells (grey arrowheads). The cytoplasm is also miRNA-30c positive. (b) Schematic overview of the miRNA-30c overexpression construct used for the generation of transgenic mice. (c) Northern blot for miRNA-30c in wildtype and transgenic littermates in line B at 8 weeks of age. U6 was used as a loading control and shows similar loading. (d) Quantification of miRNA-30c overexpression by qPCR in both transgenic lines at 4 weeks of age (N≥6). (e) MiRNA-30c expression in left en right ventricular tissue of line B at 4 weeks of age (N = 6). Error bars represent s.e.m. and * denotes a p-value ≤0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008570&req=5

pone-0096290-g001: MiRNA-30c expression in the heart and the generation of αMHC-miRNA-30c transgenic mice (miRNA-30c TG).(a) MiRNA-30c in situ hybridization on adult wildtype hearts shows expression in the nuclei of both cardiomyocytes (black arrowheads) and interstitial cells (grey arrowheads). The cytoplasm is also miRNA-30c positive. (b) Schematic overview of the miRNA-30c overexpression construct used for the generation of transgenic mice. (c) Northern blot for miRNA-30c in wildtype and transgenic littermates in line B at 8 weeks of age. U6 was used as a loading control and shows similar loading. (d) Quantification of miRNA-30c overexpression by qPCR in both transgenic lines at 4 weeks of age (N≥6). (e) MiRNA-30c expression in left en right ventricular tissue of line B at 4 weeks of age (N = 6). Error bars represent s.e.m. and * denotes a p-value ≤0.05.
Mentions: To examine the cell types expressing miRNA-30c in the heart in vivo, we performed in situ hybridization on hearts of wildtype mice using a Fam-labeled LNA probe against miRNA-30c (Figure 1a). This staining revealed expression of miRNA-30c in the nuclei of both cardiomyocytes (black arrowheads) and non-cardiomyocytes (grey arrowheads). Hybridization with the control probe displayed no signal. In addition, we found a diffuse pattern of miRNA-30c expression in the cytoplasm of cardiomyocytes which was not observed in sections treated with the control probe. The nuclear staining likely identifies pre-miRNA-30c while the cytoplasmic staining detects mature miRNA-30c. The expression of miRNA-30c in both cardiomyocytes and non-cardiomyocytes confirms previously reported in vitro studies [12], [13], which show expression in cultured neonatal myocytes and fibroblasts of the rat heart.

Bottom Line: We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy.This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level.We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function.

View Article: PubMed Central - PubMed

Affiliation: Heart Failure Research Center, Academic Medical Center, Amsterdam, The Netherlands; Interuniversitair Cardiologisch Instituut Nederland (ICIN-NHI), Utrecht, The Netherlands.

ABSTRACT
MicroRNAs (miRNAs) regulate many aspects of cellular function and their deregulation has been implicated in heart disease. MiRNA-30c is differentially expressed in the heart during the progression towards heart failure and in vitro studies hint to its importance in cellular physiology. As little is known about the in vivo function of miRNA-30c in the heart, we generated transgenic mice that specifically overexpress miRNA-30c in cardiomyocytes. We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy. Gene expression analysis of the miRNA-30c transgenic hearts before onset of the phenotype indicated disturbed mitochondrial function. This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level. Taken together these data indicate impaired mitochondrial function due to OXPHOS protein depletion as a potential cause for the observed dilated cardiomyopathic phenotype in miRNA-30c transgenic mice. We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function.

Show MeSH
Related in: MedlinePlus