Cardioprotective role of the mitochondrial ATP-binding cassette protein 1

Abstract

The mechanism by which mitochondria exert protection against oxidant stress is not clear. We recently showed that a purified mitochondrial fraction containing 5 coimmunoprecipitating proteins (succinate dehydrogenase, adenine nucleotide translocator, ATP synthase, inorganic phosphate carrier, and mitochondrial ATP-binding cassette protein 1 or mABC1) displayed mitochondrial ATP-sensitive K+-channel activity. mABC1, a member of the ABC family of proteins, is the only protein in this complex whose function is not known. A yeast homologue of mABC1 protein, Mdl1p, was recently identified to have a novel role for induction of cellular resistance to oxidant stress. Based on these observations, we hypothesized that mABC1 plays a key role in protection of myocardial cells against oxidant stress. We studied the function of mABC1 by modulating the levels of this protein in neonatal rat cardiomyocytes using various molecular techniques, followed by assessment of cell viability and measurement of mitochondrial membrane potential. RNA interference resulted in reduced mABC1 mRNA and protein levels and was associated with significantly attenuated loss of tetramethylrhodamine ethyl ester fluorescence under basal conditions and an increase in trypan blue stained cells. In contrast, adenovirally mediated expression of mABC1 resulted in protection against oxidant stress loss of mitochondrial membrane potential. These results support the notion that mABC1 protein plays a major role in cellular protection against oxidant stress, identifying mABC1 as a novel target for cardioprotective therapeutics.

Figure 1

A, Confocal images of cells transfected with rhodamine-labeled mABC1 SiRNA. Cells display the expected extranuclear punctuate fluorescence. B, Treatment of cells with mABC1 SiRNA resulted in a significant decrease in mABC1 mRNA in NRCMs. The change ranged from 40% to 95% reduction in mRNA levels. C, Cell extracts were analyzed on SDS-PAGE gels and were probed with mABC1 antibody. There was a significant reduction in mABC1 protein levels compared with nonsilencing control SiRNA-transfected cells. D, Flow cytometry of NRCMs loaded with the mitochondrial membrane potential sensitive dye TMRE. Cardiac myocytes displayed a significant loss of membrane potential when treated with mABC1 SiRNA for 48 hours (open histogram) compared with sham-transfected cells (pink histogram). E, Summary of data of NRCMs treated with mABC1 SiRNA compared with controls. There was a significant ≈40% reduction in the number of cells with high fluorescence (>10²) and a significant decrease in the number of cells with lower fluorescence (<10²). N=6 in each group.

Figure 2

Overexpression of mABC1 protein results in protection against H₂O₂-induced loss of mitochondrial membrane potential. A, Confocal image of NRCMs transduced with AdCIG-mABC1 adenovirus. Higher titers of the virus (>5 × 10⁹ plaque forming units [PFU]) resulted in higher number of cell death, ie, cytopathic effects of the virus. B, Western blot analysis of extracts of cells treated with AdCIG-mABC1 and blotted with mABC1 antibody. There was a significant increase in the levels of mABC1 protein in cells treated with the adenovirus. Longer exposure of the gel showed a band at 55 kDa in the nontransduced cells. C, Cells experienced a significant loss of mitochondrial membrane potential when exposed to 50 µmol/L H₂O₂; however, this effect was reversed by the addition of the mABC1 adenovirus.