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 ß (TGFß) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFß/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFß(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 TGFß(1) (1 ng/ml). TGFß(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 TGFß(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFß(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 TGFß(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 TGFß signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFß receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFß(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFß(1) treatment, SMAD signaling was still responsive to TGFß(1) indicated by an upregulation of SMAD7, a TGFß(1) target protein. CONCLUSION: Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFß(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFß signaling.
