Modification of the source or fate of H+ reduces Ca2+ overload and improves recovery of post-ischaemic cardiac function

Alexander S. Clanachan, Lianguo Wang & Gary D. Lopaschuk, Department of Pharmacology, University of Alberta, Edmonton, Canada, T6G2H7.

Accumulation of protons (H+) during myocardial ischaemia causes intracellular acidosis and plays a key role in ischaemia-reperfusion (I-R) injury. We have demonstrated (Liu et al., 2003) that decreasing the source of H+ by improving the coupling of glycolysis to glucose oxidation accelerates recovery from post-ischaemic acidosis, and hence lowers the potential for Ca2+ accumulation during reperfusion. Similarly, alteration of the fate of H+ by inhibition of the Na+-H+-exchanger (NHE), thereby potentially preventing N+ and Ca2+ accumulation, also enhances recovery of post-ischaemic myocardial function ( Scholz et al., 1995). However, links between the source or fate of H+, changes in intracellular Ca2+ levels (Ca2+i) and recovery of mechanical function of the post-ischaemic heart have hitherto not been established. This study tested the hypothesis that drug-induced alteration of either the source or fate of H+ will reduce post-ischaemic Ca2+i overload and enhance recovery of mechanical function.

Hearts were removed from pentobarbitone-anaesthetized (60 mg.kg-1) male Sprague Dawley rats (300-400g) and perfused in the working mode (paced at 300 beats.min-1) at 37 oC with Krebs solution containing glucose (11 mM), palmitate (1.2 mM) and insulin (100 mU.L-1). Hearts were loaded for 25 min with the fluorescence Ca2+ indicator, Indo-1AM (5 µM). Indo-1 fluorescence at 405 nm and 485 nm was measured from the epicardial surface of a 0.3 cm2 area of the left ventricular (LV) free wall using a spectrofluorometer fitted with a bifurcated fiber optic cable containing both excitation and emission bundles. Signals were acquired at 500 Hz and the ratio of Indo-1 fluorescence at 405 nm and 485 nm was calculated and used as an index of diastolic (dCa2+i) and systolic (sCa2+i) Ca 2+ levels. LV minute work (cardiac output x LV developed pressure, L.min -1.mmHg) was measured throughout the perfusions. After baseline aerobic measurements of Ca2+i and LV work, hearts were subjected to 20 min of global no-flow ischaemia at 37 oC and 30 min of working aerobic reperfusion in the absence (n=8) or presence of either dichloroacetate, an activator of glucose oxidation that inhibits H+ production (DCA, 3 mM, n=6, present during R), or cariporide, an inhibitor of NHE (CAR, 5 µM, n=6, present throughout IR).

After reperfusion, indices (mean±s.e.mean) of dCa2+i and sCa2+i in untreated hearts were increased (P<0.05) from 0.171 ± 0.003 and 0.244 ± 0.005, respectively, to 0.203 ± 0.004 and 0.278±0.004. DCA or CAR reduced (P<0.05) accumulation of both dCa2+i (to 0.186±0.005 and 0.179±0.003, respectively, P<0.05) and sCa2+i (to 0.256±0.005 and 0.252±0.003, respectively, P<0.01) during reperfusion. During reperfusion, recovery of LV work in the untreated group was 23.6±4.7% of aerobic values and was increased (P<0.05) by DCA (to 65.2±9.9%) and CAR (to 56.3±7.8%).

These data provide the first direct evidence that the beneficial cardioprotective effect on LV mechanical function elicited by either inhibition of the source of H+ production or alteration of the fate of H+ can be attributed to a reduction in Ca2+i accumulation.

Lui, Q. et al., (2003). J Am Coll Cardiol, 39 , 718-725.
Scholz, W. et al., (1995). Cardiovasc Res, 29, 260-268.