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.
Haghikia
Arash
A
Department of Cardiology and Angiology, Medical School Hannover, Hannover, Germany.
Missol-Kolka
Ewa
E
Tsikas
Dimitrios
D
Venturini
Letizia
L
Brundiers
Stephanie
S
Castoldi
Mirko
M
Muckenthaler
Martina U
MU
Eder
Matthias
M
Stapel
Britta
B
Thum
Thomas
T
Haghikia
Aiden
A
Petrasch-Parwez
Elisabeth
E
Drexler
Helmut
H
Hilfiker-Kleiner
Denise
D
Scherr
Michaela
M
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