Pivotal role of miR-448 in the development of ROS-induced cardiomyopathy.
OBJECTIVE
Nicotinamide adenine dinucleotide oxidases (NOXs) are important contributors to cellular oxidative stress in the cardiovascular system. The NOX2 isoform is upregulated in numerous disorders, including dystrophic cardiomyopathy, where it drives the progression of the disease. However, mechanisms underlying NOX2 overexpression are still unknown. We investigated the role of microRNAs (miRs) in the regulation of NOX2 expression.


RESULTS
Duchenne muscular dystrophy (DMD) was used as a model of cardiomyopathy. After screening with miRNA target prediction databases and following qRT-PCR analysis, we found drastic downregulation of miR-448-3p in hearts of mdx mice, an animal model of DMD. The downregulation correlated with overexpression of the Ncf1 gene, encoding the NOX2 regulatory subunit p47(phox). Specificity of Ncf1 targeting by miR-448-3p was validated by luciferase reporter assay. Silencing of miR-448-3p in wild-type mice had a dramatic effect on cellular and functional properties of cardiac muscle as assessed by western blotting, qRT-PCR, confocal imaging, echocardiography, and histology. Acute treatment of mice with LNA-miR-448 inhibitors led to increased Ncf1 expression, abnormally elevated reactive oxygen species (ROS) production and exacerbated Ca(2+) signalling in cardiomyocytes, reminiscent of features previously observed in dystrophic cardiac cells. In addition, chronic inhibition of miR-448-3p resulted in dilated cardiomyopathy and arrhythmia, hallmarks of dystrophic cardiomyopathy.


CONCLUSIONS
Our studies suggest that downregulation of miR-448-3p leads to the increase in the expression of Ncf1 gene and p47(phox) protein, as well as to the substantial increase in NOX2-derived ROS production. Cellular oxidative stress subsequently triggers events that finally culminate in cardiac tissue damage and development of cardiomyopathy.
Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School - Rutgers University, 185 S. Orange Avenue, Newark 07103, NJ, USA.
Sergii
S
Kyrychenko
Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School - Rutgers University, 185 S. Orange Avenue, Newark 07103, NJ, USA.
Viktoriia
V
Kyrychenko
Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School - Rutgers University, 185 S. Orange Avenue, Newark 07103, NJ, USA.
Myriam A
MA
Badr
Department of Cell Biology and Molecular Medicine, New Jersey Medical School - Rutgers University, Newark 07103, NJ, USA.
Yoshiyuki
Y
Ikeda
Department of Cell Biology and Molecular Medicine, New Jersey Medical School - Rutgers University, Newark 07103, NJ, USA.
Junichi
J
Sadoshima
Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School - Rutgers University, 185 S. Orange Avenue, Newark 07103, NJ, USA nshiroko@njms.rutgers.edu.
Natalia
N
Shirokova
D000818:Animals; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling; D002311:Cardiomyopathy, Dilated / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D004195:Disease Models, Animal; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D018101:Mice, Inbred mdx; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000150:complications / Q000235:genetics; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D020257:Ventricular Remodeling
C000610584:MIRN448 microRNA, mouse; D008562:Membrane Glycoproteins; D035683:MicroRNAs; D017382:Reactive Oxygen Species; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1
Cardiomyopathy; Dystrophin; NOX; Oxidative stress; microRNA