Ischemia time impacts on respiratory chain functions and Ca2+-handling of cardiac subsarcolemmal mitochondria subjected to ischemia reperfusion injury.

ABSTRACT

BACKGROUND: Mitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). Despite cardioplegic arrest, IR-associated cardiodepression is a major problem in heart surgery. We determined the effect of increasing ischemia time on the respiratory chain (RC) function, the inner membrane polarization and Ca2+ homeostasis of rat cardiac subsarcolemmal mitochondria (SSM). METHODS: Wistar rat hearts were divided into 4 groups of stop-flow induced warm global IR using a pressure-controlled Langendorff system 0, 15, 30 and 40 min of ischemia with 30 min of reperfusion, respectively. Myocardial contractility was determined from left ventricular pressure records (dP/dt, dPmax) with an intraventricular balloon. Following reperfusion, SSM were isolated and analyzed regarding electron transport chain (ETC) coupling by polarography (Clark-Type electrode), membrane polarization (JC1 fluorescence) and Ca2+-handling in terms of Ca2+-induced swelling and Ca2+-uptake/release (Calcium Green-5 N® fluorescence). RESULTS: LV contractility and systolic pressure during reperfusion were impaired by increasing ischemic times. Ischemia reduced ETC oxygen consumption in IR40/30 compared to IR0/30 at complex I-V (8.1 ± 1.2 vs. 18.2 ± 2.0 nmol/min) and II-IV/V (16.4 ± 2.6/14.8 ± 2.3 vs. 2.3 ± 0.6 nmol/min) in state 3 respiration (p < 0.01). Relative membrane potential revealed a distinct hyperpolarization in IR30/30 and IR40/30 (171.5 ± 17.4% and 170.9 ± 13.5%) compared to IR0/30 (p < 0.01), wearing off swiftly after CCCP-induced uncoupling. Excess mitochondrial permeability transition pore (mPTP)-gated Ca2+-induced swelling was recorded in all groups and was most pronounced in IR40/30. Pyruvate addition for mPTP blocking strongly reduced SSM swelling in IR40/30 (relative AUC, ± pyruvate; IR0/30 1.00 vs. 0.61, IR15/30 1.68 vs. 1.00, IR30/30 1.42 vs. 0.75, IR40/30 1.97 vs. 0.85; p < 0.01). Ca2+-uptake remained unaffected by previous IR. Though Ca2+-release was delayed for ≥30 min of ischemia (p < 0.01), Ca2+ retention was highest in IR15/30 (RFU; IR0/30 6.3 ± 3.6, IR 15/30 42.9 ± 5.0, IR30/30 15.9 ± 3.8, IR40/30 11.5 ± 6.6; p ≤ 0.01 for IR15/30 against all other groups). CONCLUSIONS: Ischemia prolongation in IR injury gradually impaired SSM in terms of respiratory chain function and Ca2+-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca2+-cycling and ETC function. Ischemia time should be considered an important factor influencing IR experimental data on subsarcolemmal mitochondria. Periods of warm global ischemia should be minimized during cardiac surgery to avoid excessive damage to SSMs.