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

Abstract 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 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 TGFβ1 -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 TGFβ1 (1 ng/ml). TGFβ1 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 μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ1 ‐induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ1 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 TGFβ1 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 TGFβ1 stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ1 treatment, SMAD signaling was still responsive to TGFβ1 indicated by an upregulation of SMAD7, a TGFβ1 target protein. Conclusion Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ1 by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling.