title: Transgenic overexpression of the adenine nucleotide translocase 1 protects cardiomyocytes against TGFβ1-induced apoptosis by stabilization of the mitochondrial permeability transition pore.
abstract: OBJECTIVE
  Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin-angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor β (TGFβ) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFβ/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFβ(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected.
  
  
  RESULTS
  Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFβ(1) (1 ng/ml). TGFβ(1) treatment of WT cells enhanced the number of apoptotic cells by 31.8 ± 11.7% (p<0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ(1) decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0 ± 3.7% in WT cardiomyocytes, but only by 7.2 ± 2.8% (p<0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFβ(1) in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFβ signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFβ receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFβ(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ(1) treatment, SMAD signaling was still responsive to TGFβ(1) indicated by an upregulation of SMAD7, a TGFβ(1) target protein.
  
  
  CONCLUSIONS
  Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling.
authors.affiliation: Justus-Liebig University, Institute of Physiology, Giessen, Germany. Jacqueline.Heger@ugcvr.de
authors.forename: J
authors.initials: J
authors.lastname: Heger
authors.forename: Y
authors.initials: Y
authors.lastname: Abdallah
authors.forename: T
authors.initials: T
authors.lastname: Shahzad
authors.forename: I
authors.initials: I
authors.lastname: Klumpe
authors.forename: H M
authors.initials: HM
authors.lastname: Piper
authors.forename: H-P
authors.initials: HP
authors.lastname: Schultheiss
authors.forename: K-D
authors.initials: KD
authors.lastname: Schlüter
authors.forename: R
authors.initials: R
authors.lastname: Schulz
authors.forename: G
authors.initials: G
authors.lastname: Euler
authors.forename: A
authors.initials: A
authors.lastname: Dörner
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D015870:Gene Expression; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D019076:Transgenes
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D053773:Transforming Growth Factor beta1; D000226:Mitochondrial ADP, ATP Translocases