TWIST1 regulates the activity of ubiquitin proteasome system via the miR-199/214 cluster in human end-stage dilated cardiomyopathy.
BACKGROUND
The transcription factor TWIST1 has been described to regulate the microRNA (miR)-199/214 cluster. Genetic disruption of TWIST1 resulted in a cachectic phenotype and early death of the knock-out mice. This might be connected to the activity of the ubiquitin-proteasome-system (UPS), as miR-199a has been suggested to regulate the ubiquitin E2 ligases Ube2i and Ube2g1.


METHODS
Cardiac tissue from explanted hearts of 42 patients with dilated cardiomyopathy and 20 healthy donor hearts were analysed for protein expression of TWIST1 and its inhibitors Id-1, MuRF-1 and MAFbx, the expression of miR-199a, -199b and -214, as well as the activity of the UPS by using specific fluorogenic substrates.


RESULTS
TWIST1 was repressed in patients with dilated cardiomyopathy by 43% (p=0.003), while Id1 expression was unchanged. This was paralleled by a reduced expression of miR-199a by 38 ± 9% (p=0.053), miR-199b by 36 ± 13% (p=0.019) and miR-214 by 41 ± 11% (p=0.0158) compared to donor hearts. An increased peptidylglutamyl-peptide-hydrolysing activity (p<0.0001) was observed in the UPS, while the chymotrypsin-like and trypsin-like activities were unchanged. The protein levels of the rate limiting ubiquitin E3-ligases MuRF-1 and MAFbx were up-regulated (p=0.005 and p=0.0156, respectively). Mechanistically silencing of TWIST1 using siRNA in primary rat cardiomyocytes led to a down-regulation of the miR-199/214 cluster and to a subsequent up-regulation of Ube2i.


CONCLUSIONS
The TWIST1/miR-199/214 axis is down-regulated in dilated cardiomyopathy, which is likely to play a role in the increased activity of the UPS. This may contribute to the loss of cardiac mass during dilatation of the heart.
Center for Cardiovascular Research, Charité Medical School, Berlin, Germany; Applied Cachexia Research, Department of Cardiology, Charité Medical School, Berlin, Germany.
Anna
A
Baumgarten
Claudia
C
Bang
Anika
A
Tschirner
Anke
A
Engelmann
Volker
V
Adams
Stephan
S
von Haehling
Wolfram
W
Doehner
Reinhard
R
Pregla
Markus S
MS
Anker
Kinga
K
Blecharz
Rudolf
R
Meyer
Roland
R
Hetzer
Stefan D
SD
Anker
Thomas
T
Thum
Jochen
J
Springer
D000328:Adult; D000818:Animals; D000831:Animals, Newborn; D002311:Cardiomyopathy, Dilated / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000096:biosynthesis*; D008875:Middle Aged; D009687:Nuclear Proteins / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D051879:Twist-Related Protein 1 / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism*
C547397:MIRN214 microRNA, human; D035683:MicroRNAs; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1; D025801:Ubiquitin; C531408:mirn199 microRNA, human; D046988:Proteasome Endopeptidase Complex
Cardiomyopathy; DCM; TWIST1; Ubiquitin proteasome system; microRNA