用电子探针表征缺血再灌注心肌的亚细胞钙池。

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

作者信息

Miller T W, Tormey J M

机构信息

Department of Physiology, UCLA School of Medicine 90024-1760.

出版信息

Cardiovasc Res. 1995 Jan;29(1):85-94.

PMID:7895244

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.

摘要

目的

分析全心缺血及再灌注心肌中亚细胞水平钙及其他电解质的重新分布情况，以确定再灌注损伤的可能原因。

方法

将分离的雪貂乳头肌（维持在37℃，1.2Hz刺激）通过与室内空气隔绝、去除缓冲液并暴露于恒流的水饱和95%N₂/5%CO₂气体中，使其处于缺血状态1小时。部分肌肉通过暴露于对照缓冲液中进行“再灌注”5分钟。采用电子探针微量分析技术测量亚细胞电解质含量。

结果

缺血导致细胞严重肿胀，细胞内钠含量增加一倍[从84（标准误5）mmol·kg⁻¹干重（dw）增至156（9），P<0.05]。线粒体电解质浓度普遍升高，最显著的是钙[从0.40（0.15）mmol·kg⁻¹ dw增至1.99（0.32）mmol·kg⁻¹ dw，P<0.05]。其他结构中的电解质受影响程度相对较小。再灌注导致形成两种一般类型的心肌细胞。不可逆损伤的细胞出现挛缩结节并伴有细胞外液流入。分析仅限于中度损伤的细胞，这些细胞肿胀减轻，线粒体空泡化数量不等。在中度损伤的细胞中钠含量仍然很高[167（11）mmol·kg⁻¹ dw]，镁含量从39（2）mmol·kg⁻¹ dw增至52（2）mmol·kg⁻¹ dw（P<0.05）。线粒体钙接近对照水平[0.64（0.42）mmol·kg⁻¹ dw]。连接肌浆网钙显著增加[对照细胞和再灌注细胞分别为6.56（0.59）和18.53（1.64）mmol·kg⁻¹ dw；P<0.05]，而在T小管腔或由肌浆网、T小管和游离肌浆组成的“第三区室”中钙无显著变化。“第三区室”和连接肌浆网的结果表明肌膜结合钙减少。