title: MicroRNA-223 displays a protective role against cardiomyocyte hypertrophy by targeting cardiac troponin I-interacting kinase.
abstract: OBJECTIVE
  MicroRNAs play regulatory role in cardiovascular disease. MicroRNA-223 (miR-223) was found to be expressed abundantly in myocardium. TNNI3K, a novel cardiac troponin I (cTnI)-interacting and cardiac hypertrophy related kinase, is computationally predicted as a potential target of miR-223. This study was designed to investigate the cellular and molecular effects of miR-223 on cardiomyoctye hypertrophy, focusing on the role of TNNI3K.
  
  
  METHODS
  Neonatal rat cardiomyocytes (CMs) were cultured, and CMs hypertrophy was induced by endothelin-1 (ET-1). In vivo cardiac hypertrophy was induced by transverse aorta constriction (TAC) in rats. Expression of miR-223 in CMs and myocardium was detected by real-time PCR (RT-PCR). MiR-223 and TNNI3K were overexpressed in CMs via chemically modifed sense RNA (miR-223 mimic) transfection or recombinant adenovirus infection, respectively. Cell size was measured by surface area calculation using fluorescence microscopy after anti-α-actinin staining. Expression of hypertrophy-related genes was detected by RT-PCR. The protein expression of TNNI3K and cTnI was determined by Western blots. Luciferase assay was employed to confirm the direct binding of miR-223 to the 3'UTR of TNNI3K mRNA. Intracellular calcium was measured by sensitive fluorescent indicator (Furo-2). Video-based edge detection system was employed to measure cardiomyocyte contractility.
  
  
  RESULTS
  MiR-223 was downregulated in ET-1 induced hypertrophic CMs and in hypertrophic myocardium compared with respective controls. MiR-223 overexpression in CMs alleviated ET-1 induced hypertrophy, evidenced by smaller cell surface area and downregulated ANP, α-actinin, Myh6 and Myh7 expression. Luciferase reporter gene assay showed that TNNI3K serves as a direct target gene of miR-223. In miR-223-overexpressed CMs, the protein expression of TNNI3K was significantly downregulated. MiR-223 overexpression also rescued the upregulated TNNI3K expression in hypertrophic CMs. Furthermore, cTnI phosphorylation was downregulated post miR-223 overexpression. Ad.rTNNI3K increased intracellular Ca(2+) concentrations and cell shortening in CMs, while miR-223 overexpression significantly rescued these hypertrophic effects.
  
  
  CONCLUSIONS
  By direct targeting TNNI3K, miR-223 could suppress CMs hypertrophy via downregulating cTnI phosphorylation, reducing intracellular Ca(2+) and contractility of CMs. miR-223 / TNNI3K axis may thus be major players of CMs hypertrophy.
authors: Department of Cardiology, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, Shanghai Institute of Health Sciences, Shanghai, PR China.
authors: Yao-Sheng
authors: YS
authors: Wang
authors: Jing
authors: J
authors: Zhou
authors: Kui
authors: K
authors: Hong
authors: Xiao-Shu
authors: XS
authors: Cheng
authors: Yi-Gang
authors: YG
authors: Li
mesh_terms: D020413:3' Untranslated Regions; D000185:Actinin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D006332:Cardiomegaly / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects; D019332:Endothelin-1 / Q000494:pharmacology; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009119:Muscle Contraction; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D019210:Troponin I / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D019332:Endothelin-1; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D019210:Troponin I; D000185:Actinin; D011505:Protein-Tyrosine Kinases; C584646:TNNI3K protein, rat; D002118:Calcium