Abstract
BackgroundMitochondrial 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).MethodsWistar 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).ResultsLV 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).ConclusionsIschemia 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.
Highlights
Mitochondrial impairment can result from myocardial ischemia reperfusion injury (IR)
Depression of left ventricular (LV) contractility of IR40/30 was underlined by a significant difference to IR15/30 and a b c
We focused on the integrity and function of subsarcolemmal mitochondria (SSM) after exposure of isolated rat hearts to a clinically relevant spectrum of warm global ischemia ranging from 0 to 40 min followed by a sufficiently equilibrating 30 min of reperfusion
Summary
Mitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). 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). As ischemia reperfusion (IR) injury negatively impacts on the preservation of the inner mitochondrial membrane (IMM) potential through disruption of the electron transport chain (ETC) coupling, consecutive ATP depletion and intracellular calcium overload following reduced mitochondrial calcium retention capacity determine the loss of cardiac contractility [1]. Cardiac mitochondria have long been recognized to consist of interfibrillar and subsarcolemmal populations [2] These subgroups show distinct morphological and biochemical differences, e. We sought to comprehensively determine the influence of different durations of IR on calcium homeostasis, electron transport chain coupling and IMM polarization changes in SSM in a Langendorff perfusion model of isolated rat hearts. As intermittent cardioplegic arrest and hypothermic conditions should ameliorate any ischemia-induced effects, we sought to maximize the discriminatory power of our experiments by excluding such confounders
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