No abstract

Intracellular free calcium concentration ([Ca2+]i) was measured in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4) by loading paced hearts with the 19F NMR calcium indicator, the 5,5'-difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (5FBAPTA), to an initial cytosolic concentration of approximately 120 microM. Increasing the pacing frequency raised the end-diastolic [Ca2+]i from 299 +/- 44 nM (mean +/- SEM) at 0.2 Hz to 522 +/- 54 nM at 1.0 Hz and 691 +/- 166 nM at 2.0 Hz. Raising [Ca]o from 1.8 to 7.0 mM at a pacing frequency of 1.0 Hz increased end-diastolic [Ca2+]i to 625 +/- 39 nM. In unpaced hearts perfused with diltiazem (100 microM), [Ca2+]i fell rapidly to a steady-state value of < 100 nM after 60 min. Raising [Ca]o from 1.8 to 7.0 mM had no detectable effect on resting [Ca2+]i. The time course of the [Ca2+]i transient was measured in hearts paced at 1.1 Hz and perfused with 1.8 mM [Ca]o. The peak [Ca2+]i was approximately 2 microM at approximately 150 msec after the pacing pulse, and peak developed LVP occurred at 550 msec compared with 280 msec in control hearts not loaded with 5FBAPTA. Comparisons with data obtained by other techniques, including fluorescent [Ca2+]i indicators, imply that although the end-diastolic [Ca2+]i values obtained with 5FBAPTA in beating hearts are elevated by the concentrations of intracellular 5FBAPTA required for signal detection, the changes in [Ca2+]i observed in response to experimental interventions are qualitatively consistent with previous data.

The aim of this paper is to consider the advantages of using isolated heart preparations in studies designed to investigate the effect of hypoxia or ischaemia on myocardial cells. After a brief description of the two most frequently used experimental models of perfused hearts, namely the Langendorff preparation and the working heart preparation, some of the various methods used to induce hypoxia or ischaemia are described, as well as some of the possible techniques allowing to assess metabolic alterations occurring in these pathological situations. After discussing the limitations and advantages specific to the Langendorff and working heart preparations, the suitability of isolated heart models in studies on myocardial protection is then considered. To illustrate this point, the effect of intravenous administration of the slow calcium channel blocker bepridil (5 mg X kg-1) on post-ischaemic recovery of cardiac function and metabolism after global normothermic ischaemia of the isolated heart is reported. It is concluded that isolated heart preparations allow a fine control of experimental conditions with the advantage that functional and metabolic measurements can be easily made.

The effects of cyanide on Ca 2+ exchange in isolated ventricular myocytes and on the intracellular concentrations of Ca 2+ , Na + and H + have been investigated to assess the contribution that mitochondria might play in cellular Ca 2+ metabolism. Ionic levels were measured with ion-selective electrodes. KCN (2.5 mM) inhibited a component of Ca 2+ exchange in myocytes that could be attributed to mitochondrial exchange, but was without effect on non-mitochondrial Ca 2+ exchange. NaCN (2.5 mM) caused a transient reduction of [H + ] i , [Na + ] i and [Ca 2+ ] i when applied to the superfusate bathing ventricular trabeculae or papillary muscles. The transient changes of [Na + ] i were accentuated when the preparation was exposed to a solution which would be expected to increase the cellular calcium content. The reduction of [Na + ] i which accompanies a reduction of the extracellular sodium concentration, [Na] o , was attenuated in the presence of NaCN, but the intracellular acidosis resulting from a reduction of [Na] o was unaffected by NaCN. A small, but significant, rise of [Ca 2+ ] i accompanied a reduction of [Na] o but only when NaCN was present in the superfusate. It is concluded that cyanide ions have a reasonably specific action on cardiac cellular ionic metabolism. Its primary action is to prevent mitochondrial Ca 2+ sequestration. It is postulated that a Na + /H + exchange, possibly at the sarcolemma, could account for some of the changes to sarcoplasmic ionic levels observed. In a solution of low [Na] o , it is concluded that mitochondria could sequester at least 30% of the calcium accumulated by the cell even though the sarcoplasmic [Ca 2+ ] does not exceed 0.3 μM.

Calcium influx and efflux were measured during hypoxia and on reoxygenation in the isolated arterially perfused septum of the rabbit heart. The uptake of 47Ca2+ was continuously followed with a NaI crystal and counter. The extracellular space (ECS) was measured in a similar manner with 51Cr-EDTA. Calcium efflux was recorded by collection of effluent drops after labelling with 45Ca2+. Hypoxia caused a rapid decline of developed tension followed by a rise in resting tension. The ECS increased initially but decreased as resting tension rose. 51Cr-EDTA did not have free access to the intracellular fluid. Calcium efflux did not change and calcium influx was either unchanged or reduced. On reoxygenation calcium influx increased immediately but efflux was unaltered and 51Cr-EDTA did not enter the cell. The effects of hypoxia were altered by manipulation of temperature and substrate. The recovery of mechanical function was related to the size of the calcium influx on reoxygenation. The experiments show that a rise of resting tension during hypoxia can occur in the absence of a net gain of calcium, calcium accumulation is closely associated with the extent of tissue damage, and that calcium influx on reoxygenation is probably due to a specific abnormality and not gross disruption of the cell membrane.