Abstract

Heart muscle has to work constantly throughout the life and its energy metabolism is heavily dependent on a continuous supply of oxygen. Energy metabolism must be effectively regulated to meet the demands of changing workloads in different circumstances. If the oxygen supply is interrupted, the function of the heart is easily disturbed and cells injured. Calcium metabolism is of great importance in these pathological conditions.

In this thesis respiratory regulation was studied by non-destructive optical methods in mouse heart. The myoglobin-deficient mouse was used as an experimental model to avoid the artefact caused by intracellular myoglobin. Results show that increased consumption of energy and oxygen lead to concomitant reduction of cytochrome aa₃ and oxidation of flavoproteins. This finding supports the view that cell respiration in intact myocardium is dominantly regulated at the level of the respiratory chain.

The intracellular Ca²⁺ accumulation during ischemia is one of the major causes of irreversible ischemia-reperfusion injury. Ischemic preconditioning (IPC) has been shown to protect the heart muscle significantly from ischemic damage. In this thesis Ca²⁺ accumulation during ischemia and reperfusion was studied in perfused rat heart using Fura-2 as a fluorescent Ca²⁺ indicator. As there is a significant decrease in intracellular pH during prolonged ischemia, the pH-dependency of Fura-2 signal was taken into account. It was found that IPC attenuates Ca²⁺accumulation during ischemia and this was connected to a decrease in mitochondrial membrane potential. Both IPC and the pharmacologically induced preconditioning with the mitoK_ATP opener diaxozide were shown to be associated with increased production of superoxide monitored by means of lucigenin chemiluminescence. The superoxide production correlated with the oxidation-reduction state of flavoproteins.

We also describe here a method for measuring of intracellular free Ca²⁺ in mouse heart during ischemia by simultaneous monitoring of Fura-2 and the pH probe BCECF fluorescence by means of dual wavelength excitation of both probes. The paradoxical decrease of Fura-2 fluorescence during ischemia indicating decreasing intracellular Ca²⁺ concentration was due to the pH effect on the dissociation constant of the Fura-2-Ca²⁺ complex. When the pH-dependency of Fura-2 was compensated, an extensive Ca²⁺ accumulation during ischemia was detected. Much of the previous literature on this subject must be re-evaluated because the pH-dependency of intracellular Ca²⁺ probes has been largely overlooked.