Adenine nucleotide translocator (ANT) is a protein complex in the inner membrane of mitochondria and part of the mitochondrial permeability transition pore (MPTP). It was shown that in renin-overexpressing rats ANT1 overexpression works against development of apoptosis. The question arises whether ANT1 overexpression also protects cardiomyocytes against other apoptosis inducing stimuli and whether MPTPs are involved.

To answer this question we performed studies on isolated ventricular cardiomyocytes of WT and ANT1 rats under apoptosis inducing conditions with TGFß₁ (1 ng/ml). Incubation of cardiomyocytes with TGFß₁ enhanced the number of apoptotic cells in WT from 8.8 1.9% to 13.3 2.9% (n=20, p<0.01) whereas cardiomyocytes of ANT1 rats did not show a significant increase in apoptosis (from 8.5 1.7% to 11.1 2.2%; n=20). BAX expression was already decreased under basal conditions in ANT1 rats (100.4 16.8% vs. 65.2 5.0%; n=8–12). Under stimulation with TGFß₁ bax expression increased in WT to 144.7 16.8%, whereas in ANT1 cardiomyocytes bax expression decreased to 52.0 7.7% (p<0.01; n=8–9). To analyze the involvement of MPTPs, mitochondrial calcein loss was measured by the use of calcein/cobalt method and membrane potential Dy by JC-1. After treatment of WT cells with ionomycin (5 mM) a marked loss of calcein fluorescence about 40% occurred after five minutes indicating the opening of MPTP. In cardiomyocytes of ANT1 rats drop in calcein fluorescence due to ionomycin was significantly reduced. When cardiomyocytes were incubated with TGFß₁ membrane potential in WT cardiomyocytes declined by 18.0 3.7% after 60 minutes. The decline in membrane potential in ANT1 rats under TGFß₁ was only 7.2 2.8% (p<0.05; WT n=25, ANT1 n=18). This drop in JC-1 fluorescence could be blocked when WT cells were incubated prior to TGFß₁ stimulation with cyclosporine A (200 mM), an inhibitor of MPTPs (90.7 1.5%). This indicates that the decrease in JC-1 fluorescence after TGFß₁ stimulation is due to MPTP opening.

Conclusion: 

ANT1 overexpressing adult cardiomyocytes are protected against TGFß₁ triggered apoptosis because of reduced MPTP opening and decreased bax expression.