Mechanism of preconditioning: Ionic alterations

The mechanism by which preconditioning (brief intermittent periods of ischemia and reflow) improves recovery of function and reduces enzyme release after a subsequent 30-minute period of ischemia was investigated in perfused rat hearts. Specifically, it was hypothesized that ischemia after preconditioning would result in a decreased production of H⁺ and therefore a smaller rise in [Na⁺]_i and [Ca²⁺]_i via Na⁺-H⁺ and Na⁺-Ca²⁺ exchange. To test this hypothesis we measured pH_i, [Na⁺]_i, [Ca²⁺]_i, and cell high-energy phosphates during ischemia and reflow, and we correlated this with recovery of contractile function and release of creatine kinase during reflow. ³¹P nuclear magnetic resonance (NMR) was used to measure pH_i and cell phosphates. [Na⁺]_i was measured by ²³Na NMR using the shift reagent thulium 1,4,7,10-tetraazacyclododecane-N,N,′N″,N‴- tetramethylenephosphonate to distinguish intracellular from extracellular sodium. [Ca²⁺]_i was measured by ¹⁹F NMR using hearts loaded with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid, termed 5F-BAPTA. Basal time-averaged levels of pH_i, [Na⁺]_i, and [Ca²⁺]_i were 7.07±0.08, 9.4±0.8 mM, and 715±31 nM, respectively. After 30 minutes of ischemia, in preconditioned hearts, PH_i was 6.5±0.06, [Na⁺]_i was 20.9±4.4 mM, [Ca²⁺]_i was 2.1±0.4 μM, and ATP was negligible. In untreated hearts, after 30 minutes of ischemia, pH, was 6.3±0.08, [Na⁺]_i was 26.7±3.8 mM, [Ca²⁺]_i was 3.2±0.6 μM, and ATP was undetectable. During reperfusion after 30 minutes of ischemia, preconditioned hearts had significantly better recovery of contractile function than untreated hearts (71±9% versus 36±8% initial left ventricular developed pressure), and after 60 minutes of ischemia, preconditioned hearts had significantly less release of the intracellular enzyme creatine kinase (102±12 versus 164±17 IU/g dry wt). We also found that unpreconditioned hearts arrested with 16 mM MgCl₂ (to inhibit calcium entry via calcium channels and Na⁺-Ca²⁺ exchange) before 30 minutes of ischemia recover function on reflow to the same extent as preconditioned hearts with or without magnesium arrest. Thus, preconditioning has no additional benefit in addition to magnesium arrest. In addition, in hearts that received 16 mM MgCl₂ just before the 30-minute period of ischemia, preconditioning had no effect on the rise in [Ca²⁺]_i during the 30-minute period of ischemia. These data support the hypothesis that preconditioning attenuates the increase in [Ca²⁺]_i, [Na⁺]_i, and [H⁺]_i during ischemia, most likely because of reduced stimulation of Na⁺-H⁺ and Na⁺-Ca²⁺ exchange. The data suggest that interventions that minimize ionic derangements during ischemia are associated with improved recovery of contractility and less enzyme release on reflow.