Protection of Ischaemic‐reperfused Rat Heart by Dimethylamiloride Is Associated With Inhibition of Mitochondrial Permeability Transition

Abstract

1. The aim of the present study was to assess whether protection afforded by the Na(+)/H(+) exchanger blocker dimethylamiloride (DMA) is associated with inhibition of mitochondrial permeability transition (MPT). The effects of DMA were compared with those of cyclosporine (Cs) A, an inhibitor of MPT. 2. Rat hearts were Langendorff perfused with Krebs'-bicarbonate medium containing 10 mmol/L glucose and were subjected to 25 min no-flow global ischaemia and 30 min reperfusion in the presence or absence of 10 micromol/L DMA or 0.2 micromol/L CsA. Cell viability was measured using tetrazolium stain. The MPT was determined by loading hearts with 2-deoxy-[(3)H]-glucose (2DG), which enters mitochondria only during MPT. Total heart 2DG content as an estimation of the extent of tissue damage was also measured. To assess whether DMA has any direct effect on glycolysis, a cell-free heart extract containing all the glycolytic enzymes was used. 3. Dimethylamiloride improved functional recovery (rate-pressure product) from 24 +/- 7 to 68 +/- 11% (P < 0.01) at reperfusion end, attenuated the increase in left ventricular end-diastolic pressure (from 29 +/- 7 to 6 +/- 3% 10 min after reperfusion onset; P < 0.01), improved cell viability (from 21.2 +/- 6.6 to 69.6 +/- 7.1% at reperfusion end; P < 0.05) and lessened lactate accumulation at the end of ischaemia (119 +/- 15 vs 163 +/- 14 micromol/g dry weight; P < 0.05). Dimethylamiloride limited MPT: 2DG mitochondrial entrapment, being 33.1 +/- 14.2 and 96.3 +/- 14.0 at reperfusion end in the treated and control hearts, respectively (P < 0.05), and concomitantly raised total 2DG content (51.3 +/- 4.4 vs 86.8 +/- 1.7 x 10(3) d.p.m./g wet weight in control and treated groups, respectively; P < 0.05). Cyclosporine A improved functional recovery and attenuated the amplitude of ventricular diastolic pressure in ischaemic-reperfused hearts. It also reduced mitochondrial entrapment (67.3 +/- 7.7%; P < 0.05 vs control) and increased total cell 2DG content (162.3 +/- 1.3 x 10(3) d.p.m./g wet weight; P < 0.01 vs control) at the end of reperfusion. Dimethylamiloride did not affect glucose consumption and lactate production in the cell-free heart extract. 4. In conclusion, DMA protects against the noxious effects of ischaemia-reperfusion and inhibits MPT, coinciding with present and previous findings concerning the effects of CsA. Dimethylamiloride also diminished lactate accumulation, although it did not exhibit any direct effect on glycolysis. These data suggest that blockade of Na(+)/H(+) exchange by DMA attenuates the extent of MPT in ischaemic-reperfused rat heart.

Mitochondrial Permeability Transition Pore Opening as a Promising Therapeutic Target in Cardiac Diseases

“…A delayed pH i recovery has been shown to correlate with inhibition of MPTP opening in IR (Kerr et al, 1999). We and others Prendes et al, 2008) demonstrated that cardioprotection induced by NHE-1-specific inhibitors against IR was associated with inhibition of MPTP opening. Hearts treated with NHE-1 inhibitors demonstrated reduced myocyte injury as shown by decreased low LDH release and greater recovery of cardiac function at reperfusion .…”

“…A detailed discussion of the main principles and advantages/ disadvantages of both methods mentioned above has been provided previously (Halestrap et al, 2004;Javadov and Karmazyn, 2007). The DOG-method is used widely by various groups working in experimental cardiology to study the protective effects of pharmacological and conditional inter- ventions on MPTP opening during IR and HF (Javadov et al, 2005;Ciminelli et al, 2006;Prendes et al, 2008).…”

A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection

“…The myocytes endeavour to restore intracellular pH by activating NHE1 and this increases intracellular Na + concentration allowing Ca 2+ to enter by reversal of NHE1 [190,191]. Indeed, amiloride derivatives such as cariporide that inhibit NHE1 have been shown to protect the heart from reperfusion injury [192] and, using the Hot-DOG technique, to reduce MPTP opening in situ [193][194][195]. Another protective mechanism that may involve the maintenance of a more acid pH during reperfusion is 'post-conditioning' in which very brief (10 s) intermittent ischaemic periods are included within the first few minutes of reperfusion [196][197][198].…”

Protective Effects of Co-Administration of Gallic Acid and Cyclosporine on Rat Myocardial Morphology Against Ischemia/Reperfusion

“…Heart rate and perfusion pressure were monitored continuously ( 9 ). CsA (0.2 µM) was dissolved in ethanol and added to Krebs-Henseleit solution 13 minutes before the induction of ischemia and continued during reperfusion ( 11 , 12 ). After the required period of I/R, the heart was removed from the cannula, then preserved in 10% formalin for histological studies.…”