Subcellular calcium pools of ischaemic and reperfused myocardium characterised by electron probe.

ABSTRACT

OBJECTIVE: The subcellular redistribution of calcium and other electrolytes was analysed in myocardium subjected to global ischaemia and reperfusion in order to establish possible causes of reperfusion injury. METHODS: Isolated ferret papillary muscles (maintained at 37 degrees C, 1.2 Hz stimulation) were exposed to ischaemic conditions for 1 h by isolating from room air, removing buffer, and exposing to a constant flow of water-saturated 95% N2/5% CO2 gas. Some muscles were "reperfused" for 5 min by exposing to control buffer. Electron probe microanalysis was used to measure subcellular electrolyte content. RESULTS: Ischaemia caused severe swelling and doubling of cell sodium [84(SEM 5) to 156(9) mmol.kg-1 dry weight (dw), P < 0.05]. Mitochondrial electrolyte concentrations were generally increased, most notably calcium [0.40(0.15) to 1.99(0.32) mmol.kg-1 dw, P < 0.05]. Electrolytes in other structures were more moderately affected. Reperfusion resulted in two general myocyte populations. Irreversibly injured cells exhibited contracture knots and an influx of extracellular fluid. Analysis was restricted to moderately injured cells, which had reduced swelling and varying numbers of vacuolated mitochondria. In the moderately injured cells, sodium remained high [167(11) mmol.kg-1 dw] and magnesium had increased from 39(2) to 52(2) mmol.kg-1 dw (P < 0.05). Mitochondrial calcium was near control levels [0.64(0.42) mmol.kg-1 dw]. Junctional sarcoplasmic reticular calcium significantly increased [6.56(0.59) to 18.53(1.64) mmol.kg-1 dw in control and reperfused cells, respectively, P < 0.05], while calcium had not changed significantly in T tubule lumen or "3rd compartment", composed of sarcoplasmic reticulum, T tubules, and free sarcoplasm. "3rd compartment" and junctional sarcoplasmic reticulum results suggest that sarcolemma bound calcium had decreased. CONCLUSIONS: Surviving myocytes had no change in total calcium, but showed a large degree of calcium redistribution. Mitochondria accumulated calcium during ischaemia, but released it upon reperfusion. Sarcoplasmic reticulum in reperfused cells appeared functional and may have helped maintain physiological [Ca2+]i by storing calcium released from mitochondria and the sarcolemma. Mitochondrial and sarcolemmal damage are proposed as critical factors in reperfusion injury.