Signal transducer and activator of transcription 3-mediated regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a key role for ubiquitin-conjugating enzymes.
OBJECTIVE
Mice with a cardiomyocyte (CM)-restricted knockout of signal transducer and activator of transcription 3 (STAT3-KO) develop spontaneous heart failure. We investigated the impact of STAT3-mediated regulation of microRNAs for pathophysiological alterations in the heart.


RESULTS
MicroRNAchip and qRT-PCR analysis revealed elevated cardiac expression of miR-199a in STAT3-KO mice. Lentiviral shRNA-mediated STAT3-knock-down in neonatal rat CMs markedly increased miR-199a promoter activity and miR-199a levels indicative of a suppressive effect of STAT3 on miR-199a transcription. Up-regulated miR-199a in CM by pre-miR-199a transfection (pre-miR-199a-CM) reduced expression of components of the ubiquitin-proteasome system (UPS), i.e. the ubiquitin-conjugating enzymes Ube2g1 (mRNA and protein) and Ube2i (protein). Pre-miR-199a-CM or CM with siRNA-mediated down-regulation of Ube2i and Ube2g1 (siRNA-Ube2i/2g1-CM) displayed massive down-regulation of α- and β-myosin heavy chain expression associated with disrupted sarcomere structures. In addition, protein arginine methyltransferase I (PRMT-I) expression and asymmetric dimethylarginine (ADMA) synthesis were increased in pre-miR-199a-CM or in siRNA-Ube2i/2g1-CM. Increased ADMA in cell culture supernatant (SN) from pre-miR-199a-CM or siRNA-Ube2i/2g1-CM lowered nitric oxide (NO) bioavailability of rat cardiac endothelial cells while lowering ADMA concentration in CM SNs by the PRMT inhibitor arginine methyltransferase inhibitor 1 (AMI-1) (100 µM) improved NO bioavailability. In STAT3-KO hearts Ube2i and Ube2g1 expression were markedly reduced. Human terminal failing hearts harbouring low STAT3 protein levels displayed increased miR-199a levels and decreased Ube2g1 expression.


CONCLUSIONS
This study identifies a novel pathophysiological circuit in the heart between reduced STAT3 protein levels, increased miR-199a expression, and subsequent impairment of the UPS that disrupts CM sarcomere structure and impairs via the release of ADMA endothelial cell function.
Department of Cardiology and Angiology, Medical School Hannover, Hannover, Germany.
Arash
A
Haghikia
Ewa
E
Missol-Kolka
Dimitrios
D
Tsikas
Letizia
L
Venturini
Stephanie
S
Brundiers
Mirko
M
Castoldi
Martina U
MU
Muckenthaler
Matthias
M
Eder
Britta
B
Stapel
Thomas
T
Thum
Aiden
A
Haghikia
Elisabeth
E
Petrasch-Parwez
Helmut
H
Drexler
Denise
D
Hilfiker-Kleiner
Michaela
M
Scherr
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D015536:Down-Regulation; D042783:Endothelial Cells / Q000502:physiology*; D004730:Endothelium, Vascular / Q000502:physiology; D006333:Heart Failure / Q000201:enzymology* / Q000503:physiopathology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000502:physiology; D051381:Rats; D050796:STAT3 Transcription Factor / Q000378:metabolism / Q000502:physiology*; D044763:Ubiquitin-Conjugating Enzymes / Q000502:physiology*; D015854:Up-Regulation
C552425:MIRN199 microRNA, rat; D035683:MicroRNAs; D050796:STAT3 Transcription Factor; C018524:N,N-dimethylarginine; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D044763:Ubiquitin-Conjugating Enzymes