Key Points
- To determine whether myocardial intracellular sodium accumulation during ischemia and reperfusion is driven by sodium leak mechanisms—including Na/H exchange and Na-K-2Cl cotransport—rather than impaired Na-K pump efflux alone.
- Perfused isolated rabbit hearts (Langendorff model; n=3 per group) with oxygenated buffer during baseline, 65 minutes of ischemia, and 30 minutes of reperfusion.
- Measured intracellular sodium (Nai) and intracellular calcium ([Ca]i) using nuclear magnetic resonance spectroscopy under nominally potassium-free conditions with 1 mM ouabain to eliminate Na-K pump-mediated efflux.
- Assessed sodium uptake and efflux pathways by administering 1 mM amiloride (Na/H exchange inhibitor) or 20 µM bumetanide (Na-K-2Cl cotransport inhibitor).
- In K-free perfusate with ouabain, ischemia increased Nai from 20.2 ± 0.5 to 79.1 ± 5.3 meq/kg dry wt (ΔNai ≈ 59), whereas adding amiloride blunted this rise to 16.3 ± 0.9 to 44.7 ± 5.1 meq/kg dry wt (ΔNai ≈ 28).
- Addition of bumetanide yielded an ischemic Nai increase from 22.5 ± 3.9 to 83.8 ± 13.9 meq/kg dry wt (ΔNai ≈ 61) and completely suppressed Nai clearance during reperfusion (P < 0.05 vs bumetanide-free controls).
- Bumetanide significantly elevated [Ca]i during both ischemia and reperfusion compared to bumetanide-free conditions (P < 0.05), consistent with decreased driving force for sodium/calcium exchange.
Structured PICO
PPopulationIsolated Langendorff-perfused rabbit hearts (n=3 per protocol)
IInterventionAmiloride (1 mM) or bumetanide (20 microM) added to nominally K-free perfusate containing 1 mM ouabain
CComparatorNominally K-free perfusate containing 1 mM ouabain without amiloride or bumetanide
OOutcomeMyocardial intracellular Na (Nai) and intracellular Ca concentration ([Ca]i) during ischemia and reperfusion measured using nuclear magnetic resonance spectroscopysurrogate
Inhibition of Na-K-2Cl cotransport with bumetanide during ischemia and reperfusion increases intracellular Na and Ca, suggesting the cotransporter contributes to Na efflux during reperfusion.