MRP1/GS‐X pump ATPase expression: is this the explanation for the cytoprotection of the heart against oxidative stress‐induced redox imbalance in comparison to skeletal muscle cells?

Abstract

Striated muscle activity is always accompanied by oxidative stress (OxStress): the more intense muscle work and/or its duration, the more a redox imbalance may be attained. In spite of cardiac muscle functioning continuously, it is well known that the heart does not suffer from OxStress-induced damage over a broad physiological range. Although the expression of antioxidant enzymes may be of importance in defending heart muscle against OxStress, a series of combined antioxidant therapeutic approaches have proved to be mostly ineffective in avoiding cellular injury. Hence, additional mechanisms may be involved in heart cytoprotection other than antioxidant enzyme activities. The strong cardiotoxic effect of doxorubicin-induced cancer chemotherapy shed light on the possible role for multidrug resistance-associated proteins (MRP) in this context. Muscle activity-induced 'physiological' OxStress enhances the production of glutathione disulfide (GSSG) thus increasing the ratio of GSSG to glutathione (GSH) content inside the cells, which, in turn, leads to redox imbalance. Since MRP1 gene product (a GS-X pump ATPase) is a physiological GSSG transporter, adult Wistar rats were tested for MRP1 expression and activity in the heart and skeletal muscle (gastrocnemius), in as much as the latter is known to be extremely sensitive to muscle activity-induced OxS. MRP1 expression was completely absent in skeletal muscle. In contrast, the heart showed an exercise training-dependent induction of MRP1 protein expression which was further augmented (2.4-fold) as trained rats were challenged with a session of acute exercise. On the other hand, inducible expression of the 70-kDa heat shock protein (HSP70), a universal marker of cellular stress, was completely absent in the heart of sedentary and acutely exercised rats, whereas skeletal muscle showed a conspicuous exercise-dependent HSP70 expression, which decreased by 45% with exercise training. This effect was paralleled by a 58% decrease in GSH content in skeletal muscle which was even higher (an 80%-fall) after training thus leading to a marked redox imbalance ([GSSG]/[GSH] raised up to 38-fold). In the heart, GSH contents and [GSSG]/[GSH] ratio remained virtually unchanged even after exercise challenges, while GS-X pump activity was found to be 20% higher in the heart related to skeletal muscle. These findings suggest that an intrinsic higher capacity to express the MRP1/GS-X pump may dictate the redox status in the heart muscle thus protecting myocardium by preventing GSSG accumulation in cardiomyocytes as compared to skeletal muscle fibres.

Direct effects of doxorubicin on skeletal muscle contribute to fatigue

“…However, it did not reduce the DOX-induced increase in calcium response. Alternatively, DOX has been described to reduce GSH levels due to an extensive extrusion of GSH-conjugated DOX by MRPs (Krause et al, 2007). N-acetyl cysteine supplementation has been described to be capable of restoring GSH stores (Kelly, 1998).…”

“…However, results obtained are not completely conclusive (Singal et al, 1997). It has been described that DOX is extruded from the cardiac myocyte conjugated to GSH by multidrug resistance proteins (MRPs) (Krause et al, 2007). Extensive extrusion of DOX subsequently induces GSH depletion.…”

Caspase-dependent and -independent suppression of apoptosis by monoHER in Doxorubicin treated cells

“…In addition, cardiomyocytes were reported to have intrinsically low levels of antioxidant enzymes that may explain the potent protective function of monoHER in these cells (Hrdina et al, 2000). Also high activity of the MRP1/ GS-X pump in these cells was reported (Krause et al, 2007), which may export DOX as glutathione-Sconjugate out of the cells, thereby modifying its cytotoxic effects. However, previously we found that monoHER did not affect DOX levels in cardiac cells in mice or influence the biodistribution of DOX (Abou El Hassan et al, 2003b).…”

Physiological concentrations of interleukin-6 directly promote insulin secretion, signal transduction, nitric oxide release, and redox status in a clonal pancreatic β-cell line and mouse islets

“…As GSH is the most important antioxidant systems that b-cells use against redox-threatening situations (for instance, in immunoinflammatory situations triggered by inflammatory cytokines (Krause et al 2007)), BRIN-BD11 cells were seeded into six-well plates (2!10 6 cells/well), allowed to adhere overnight, and then washed with PBS after which they were incubated in fresh media, containing 11 . 1 mM D-glucose and 2 mM L-glutamine, in the presence or absence of sublethal pro-inflammatory cytokine cocktail with different concentrations of L-arginine (above).…”

“…Afterward, cells were rinsed twice with PBS and disrupted in 200 ml of 5% (w/v) metaphosphoric acid on ice. After centrifugation (14 000 g, 5 min at room temperature), cell lysates were spectrophotometrically (415 nm) assayed on a temperature-controlled microplate reader (Molecular Devices SpectraMax Plus 384) by a modification of the 5,5 0 -dithiobis(2-nitrobenzoic acid) (DTNB)/glutathione disulphide (GSSG) reductase recycling method, using the N-ethylmaleimide conjugating technique for GSSG sample preparation (Krause et al 2007). Samples (10 ml) were assayed in 105 ml final volume in 96-well polystyrene plates at 37 8C in the presence of 10 mM DTNB, 0 .…”