Resting and end-diastolic [Ca2+]i measurements in the Langendorff-perfused ferret heart loaded with a 19F NMR indicator.

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.

Characterization and localization of endothelin receptor subtypes in the human atrioventricular conducting system and myocardium.

The characterization and localization of endothelin A (ETA) and endothelin B (ETB) receptors have been determined in tissue sections of the human atrioventricular conducting system, surrounding regions of atrial and ventricular myocardium, and the left ventricular free wall by use of radioligand binding, polymerase chain reaction, and in situ hybridization. Selective ETA (BQ123) and ETB (BQ3020) compounds in conjunction with [125I]endothelin-1 revealed the presence of ETA and ETB receptors in the left ventricular free wall (BQ123: 57 +/- 5% ETA, 43 +/- 2% ETB, n = 3; BQ3020: 67 +/- 3% ETA, 33 +/- 3% ETB, n = 3). Autoradiography using [125I]endothelin-1 in the absence or presence of BQ3020, BQ123, or endothelin-1 showed ETA and ETB receptors localized to atrial and ventricular myocardium, the atrioventricular conducting system, and endocardial cells. There was a higher proportion of ETB receptors in the atrioventricular node and the penetrating and branching bundles of His than in the surrounding interventricular and interatrial septa (p < 0.0001). There was a lower density of ETB receptors in the interventricular septum compared with the interatrial septum and the atrioventricular conducting system (p = 0.009) and a lower density of ETA receptors in the atrioventricular conducting system compared with interatrial and interventricular septa (p = 0.008). Isolated right atrial myocytes showed a higher proportion of ETA receptors (91 +/- 12%, n = 3). Amplification of left ventricular free wall cDNA by polymerase chain reaction revealed the presence of ETA and ETB receptor mRNA. mRNA for both subtypes was detected in isolated atrial myocytes. In situ hybridization showed ETA and ETB receptor mRNA localization to atrial and ventricular myocardium, the atrioventricular conducting system, and endocardial cells. These studies demonstrate the presence of ETA and ETB receptors in human myocardium and the atrioventricular conducting system.

The contribution of mitochondrial calcium ion exchange to relaxation of tension in cardiac muscle.

The possible contribution of mitochondrial Ca2+ accumulation and release to contractile phenomena has been investigated. Two intracellular fractions of Ca2+ sequestration can be identified in cardiac myocytes, one ascribed to mitochondria. Two modes of Ca2+ transport exist within the mitochondrial fraction, one dependent upon mitochondrial respiration and the other upon extramitochondrial [Na+]. Experiments with trabeculae show that under appropriate conditions, the rate of relaxation and the amount of tension developed is dependent on these two modes of Ca2+ transport. A model is presented quantifying the contribution of the mitochondria to relaxation.

Non-mitochondrial calcium ion regulation in rat ventricular myocytes.

Ca2+ exchange has been measured in a suspension of rat ventricular myocytes treated with digitonin or saponin to render the sarcolemma permeable to small molecules and ions. Two fractions of exchange were identified, one that was attributed to the mitochondrial component of the cell and the other to a non-mitochondrial fraction. Mitochondrial Ca2+ uptake was blocked by sodium azide and depended on respiratory substrates whereas non-mitochondrial uptake occurred independently of these molecules but was dependent on ATP and creatine phosphate. Non-mitochondrial Ca2+ uptake could be induced at a Ca2+ concentration below 1 microM and the initial rate increased with concentration up to 100 microM. Uptake could be reversed by sulmazole (a caffeine-like substance) and this reversal in turn inhibited by ryanodine. These properties suggest that the major locus for non-mitochondrial Ca2+ exchange is at the sarcoplasmic reticulum. Ca2+ exchange could be modulated by a number of agents, including carnosine, but was unaffected by others, including Na+, inositol trisphosphate and cyclic AMP. A kinetic model of the data is presented, which incorporates similar data of Ca2+ uptake into the mitochondrial fraction. The rates of Ca2+ exchange measured in these experiments suggest that these two components of the cell can reduce the sarcoplasmic Ca2+ concentration rapidly enough to account for the observed transient nature of the isometric twitch. Furthermore, it is suggested that both non-mitochondrial and mitochondrial fractions of the cell could significantly contribute to tension relaxation in rat cardiac muscle.

The effects of cyanide on intracellular ionic exchange in ferret and rat ventricular myocardium.

The effects of cyanide on Ca2+ exchange in isolated ventricular myocytes and on the intracellular concentrations of Ca2+, Na+ and H+ have been investigated to assess the contribution that mitochondria might play in cellular Ca2+ metabolism. Ionic levels were measured with ion-selective electrodes. KCN (2.5 mM) inhibited a component of Ca2+ exchange in myocytes that could be attributed to mitochondrial exchange, but was without effect on non-mitochondrial Ca2+ exchange. NaCN (2.5 mM) caused a transient reduction of [H+]i, [Na+]i and [Ca2+]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 [Ca2+]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 Ca2+ 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 [Ca2+] does not exceed 0.3 microM.

The isolated perfused rat heart: a model for studying myocardial hypoxia or ischaemia.

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.

Calcium out of control.

The accumulation of calcium during myocardial hypoxia or ischaemia followed by reoxygenation or reperfusion is related to the development of cell necrosis and may be an important causal mechanism. Influx of calcium is a late event during hypoxia but occurs abruptly on reoxygenation or reperfusion. On reoxygenation calcium influx is not altered by nifedipine or quiescence but can be prevented by nickel (3 mM), cyanide (5 mM) or FCCP (10(-6) M). The extracellular marker 51Cr-EDTA does not enter the intracellular fluid on reoxygenation but can when the cell membrane is disrupted by a detergent, Brij'35, or the calcium paradox. The results suggest that the uptake of calcium on reoxygenation or reperfusion is related to the reintroduction of oxygen and caused by an increased calcium influx down the concentration gradient. The flux is not through the slow calcium channel and is not due to disruption of the membrane. The effects of CN- and FCCP and the unaltered calcium efflux suggest that the major part of the calcium uptake is stored in intracellular compartments and is not located in the intracellular fluid.

Calcium exchange in rabbit myocardium during and after hypoxia: effect of temperature and substrate.

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.

Influence of contractile state on the size of the extracellular space in isolated ventricular myocardium.

A method is described with which the extracellular space of isolated ventricular muscle can be measured accurately and continuously in the same muscle. The size of the extracellular space is shown to vary with the contractile state of the myocardium. Interventions such as quiescence, manganese and acidosis reduce myocardial contractility and increase the size of the extracellular space. Barium, ouabain and hypoxia cause contracture and reduce the size of the extracellular space. These changes should be taken into account when measuring intracellular electrolytes in isolated ventricular preparations.