Transgenic overexpression of the adenine nucleotide translocase 1 protects cardiomyocytes against TGFβ1-induced apoptosis by stabilization of the mitochondrial permeability transition pore

Aims: 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 beta (TGF beta) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGF beta/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGF beta(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved. and SMAD signaling pathway is affected. Methods and results: Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGF beta(1) (1 ng/ml). TGF beta(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 1 mu M cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 over-expression on TGF beta(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGF beta(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 TGF beta(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 over-expression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGF beta signaling pathway. Interestingly. ANT1-transgenic cardiomyocytes expressed less TGF beta receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGF beta(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGF beta(1) treatment, SMAD signaling was still responsive to TGF beta(1) indicated by an upregulation of SMAD7, a TGF beta(1) target protein. Conclusion: Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGF beta(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGF beta signaling. (c) 2012 Elsevier Ltd. All rights reserved.