The effect of ischemia and reperfusion on mitochondrial contact sites in isolated rat hearts.

Contact sites may be described as energy channels between the mitochondria and the cytosol, created by fusion of the inner and the outer mitochondrial membranes, and their number depends highly on the energy state of the cell. The aim of the present study was to examine the early changes of ischemia and reperfusion on the number of mitochondrial contact sites. Therefore isolated rat hearts were subjected to short periods of ischemia followed by reperfusion. The left ventricular pressure (LVP), the contractility (dP/dtmax) and the heart rate were measured. The number of contact sites was morphometrically evaluated. As the flow was stopped, LVP, dP/dtmax and HR declined rapidly and became undetectable after 2 min of ischemia. The number of contact sites fell to a minimum after 10 min of ischemia after an initial increase (1 min of ischemia). A 15 min ischemic period resulted in a high number of contact sites which decreased again after 20 min of ischemia. Reperfusion after 2 min of ischemia caused an immediate functional recovery and a high presence of contact sites. After 15 min of reperfusion, all values returned to control values. Reperfusion after 10 min of ischemia resulted in a slow recovery of the number of contact sites and after 15 min of ischemia the number of contact sites remained low upon reperfusion. We may conclude that mitochondria lose the ability to form contact sites after more than 15 min of ischemia and this might be a first indication of irreversible injury.

The effect of calcium on mitochondrial contact sites: a study on isolated rat hearts.

Mitochondrial contact sites are dynamic structures created by fusion of the inner and outer mitochondrial membranes. Stimulation of the metabolism results in an increase of the number of contact sites. Functionally, it is shown that mitochondrial creatine kinase (Mi-CK) is active in contact sites and therefore, Mi-CK cytochemistry was performed (using a tetrazolium salt) to improve the visibility of the contact sites. As calcium is involved as an intracellular messenger of hormonal stimulation, the effect of increasing extracellular calcium concentrations on the number of contact sites was investigated. Therefore, isolated rat hearts were perfused with Krebs-Henseleit buffers differing in their calcium content. During the perfusions the heart function was evaluated and at the end of each experiment, the hearts were processed for Mi CK cytochemistry and the number of contact sites was expressed as the ratio of surface densities contact sites to mitochondrial membranes (Ss). At 2.2 mM calcium perfusion, the physiological parameters and the Ss reached a maximum. This was in contrast to the 0.6 and the 3.6 mM of calcium perfusions whereby both the physiological values and the Ss were decreased. Treatment with noradrenaline in vivo, as was done in previous studies or perfusion with 2.2 mM of calcium ends up with similar values for Ss. From these results, it could be suggested that there might be a link between calcium, heart function and the formation of Mi CK active contact sites.

Ultrastructural localisation of carnitine acetyltransferase activity in mitochondria of rat myocardium.

The acetyl CoA/CoA ratio is an important regulating factor of beta-oxidation in mitochondria and hence of energy production in the myocardium. Carnitine acetyltransferase provides one of the control mechanisms for this ratio during changing energy demand in the heart muscle, possibly by buffering the CoA and carnitine concentration for sustained beta-oxidation. In search for a possible correlation between the activity of this enzyme and ultrastructural changes in heart mitochondria, carnitine acetyltransferase was cytochemically localised in rat myocardium, brought into different metabolic states. In this work we confirm previous observations, namely the formation of contact sites between inner and outer mitochondrial membranes upon catecholaminergic stimulation of the myocardium. It is further shown that this contact site formation might be a prerequisite for carnitine acetyltransferase to demonstrate enzymatic activity and hence control of beta-oxidation in myocardial mitochondria.

Increased extracellular calcium concentrations in the isolated rat heart: effects on mitochondrial contact site formation.

In our previous study, mitochondrial creatine kinase (Mi-CK) activity is localised cytochemically in heart tissue. The activity was found to be exclusively localised in mitochondrial contact sites and the surface density of Mi-CK was increased upon myocardial stimulation (Biermans et al., 1989). As calcium is involved as an intracellular messenger of stimulation, we compared hearts noradrenaline-stimulated in vivo with isolated hearts perfused with buffers containing different calcium concentrations on the surface density Mi-CK.

Ultrastructural localisation of creatine kinase activity in the contact sites between inner and outer mitochondrial membranes of rat myocardium.

The mitochondrial isoenzyme of creatine kinase, together with the ADP/ATP translocase, most probably belongs to a functional multi-enzyme complex located on the inner mitochondrial membrane. The outer membrane is a necessary constituent of this microcompartment. On the other hand, electron microscopic visualisation demonstrated the formation of contact sites between inner and outer mitochondrial membranes as a reaction to variations of the energy metabolism. In search for a possible correlation between these biochemical and morphological phenomena, rat myocardia were brought into the required energy state by stimulation through catecholaminergic mechanisms or adjusted perfusion with amytal. Subsequently, creatine kinase was cytochemically localised. Creatine kinase activity is demonstrated in membrane contacts between inner and outer mitochondrial membranes. The extent of contact sites and creatine kinase activity depends on the metabolic state as shown by morphometric analysis of the surface density of cytochemical reaction product. This surface density diminishes drastically after inhibiting the metabolic activity with amytal. It is concluded that these contact sites are dynamic micro-environments in which the active site of creatine kinase, oxidative phosphorylation and ADP/ATP transport interact during basal and stimulated metabolism.

A simplified 'sandwich' technique for in situ embedding and perpendicular sectioning of monolayer cultures of human skin fibroblasts.

In the processing of cell cultures, grown as a monolayer in tissue culture dishes for electron microscopy, the sectioning of the monolayer is an essential step. The monolayer can be sectioned either parallel or perpendicular to the plane of growth. Several methods for the perpendicular way of sectioning have already been described. We propose a simplified method in which the monolayer is sandwiched between two layers of resin, one of which is a prepolymerized block, the other being a layer of resin, applied at a second stage. Sectioning of this 'flat embedded' specimen yields thin sections perpendicular to the plane of growth of the monolayer without elaborate orientating procedures. The advantage of this procedure is that it can be done using only routine embedding techniques, avoiding special materials or complex manipulations. This sandwich technique provides an excellent mechanical fixation of the monolayer and protects it against external damage.

Immunocytochemical investigation of native matrix granules of the rat heart mitochondrion.

We have examined the ultrastructure and the protein content of native matrix granules (NMG) in rat heart mitochondria, by postembedding immunocytochemistry. Cytochrome c oxidase was found to be present in these granules. It is believed that these granules contain incomplete inner mitochondrial membrane fractions, which can be incorporated in the membrane after stimulation of the metabolism.