Heart muscle mitochondria with satisfactory functional parameters of oxidative phosphorylation and with morphologically intact structure were isolated from canine myocardium employing a modified KEA-medium (0.18 M KCl, 10 mM EDTA, 0.5% bovine serum albumin, pH 7.1) according to Sordahl and Schwartz (1). The functional behaviour of mitochondria was investigated after different durations of in situ ischemia (cardioplegia, 15 degrees C) and correlated with metabolic findings. During ischemia the following changes were seen: 1. Successive reduction of electron flow. 2. Relatively small impairment of phosphorylation efficiency. 3. Less damage of FAD- than NAD-catalyzed oxidative phosphorylation. 4. A marked increase of electron flow and thus recovery of phosphorylation rate even after longer ischemic periods by addition of cytochrome c. As important factors of accelerating mitochondrial impairment during ischemia the myocardial ATP decrease, the lactate and H+-activity increase are discussed.
The functional behaviour of membrane systems of the cardiac cell during oxygen deficiency was analyzed and the alterations were related to the metabolic state of the tissue as an index of injury. 1. The retention function of the cell membrane for proteins. With increasing energy deficiency the cardiac sarcolemma loses its ability to retain macromolecules (myoglobin, enzymes) within the cell. Close correlations exist between protein release and oxygen supply as well as ATP content of the tissue. 2. Function of isolated mitochondria after ischemia. In parallel with a strong impairment of oxidative phosphorylation (decrease of QO2, RCI values, phosphorylation rates) the Ca++-transporting activity of mitochondria is continuously depressed with decreasing myocardial ATP. 3. Function of isolated sarcoplasmic reticulum after ischemia. With breakdown of high energy phosphates during ischemia rate and extent of Ca++ binding with decrease markedly.
Using a DW 2 dual wavelength spectrophotometer (AMINCO) and murexide as a Ca2+ sensitive indicator it is shown that guinea-pig heart mitochondria can release accumulated Ca2+ without the influence of non-physiological material. The main factor which decides if accumulated Ca2+ is spontaneously released is the Ca2+/mitochondrial-protein ratio. Under appropriate assay conditions a critical intramitochondrial Ca2+ concentration is reached. Increasing this concentration leads to Ca2+-release.
Guinea-pig and dog heart mitochondria were isolated in a KEA-medium. Ca2+-transport across mitochondrial membranes was measured continuously with an Aminco Dual-Wavelength-Spectrophotometer and murexide as a Ca2+-sensitive indicator. Ischemia was produced by cardioplegia at 15 degrees C according to Bretschneider. Guinea-pig heart mitochondria as well as mitochondria from dog heart show a spontaneous Ca2+-release without nonphysiological influence. Addition of 3.5 M Na+ can induce a very quick release of Ca2+ taken up by heart mitochondria. This release is different from that occurring spontaneously. Progressive ischemia results in a marked depression of Ca2+-uptake and spontaneous Ca2+-release.
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