Electric currents applied during refractory period enhance contractility and systolic calcium in the ferret heart

We investigated the mechanism of positive inotropism of electric currents applied during the absolute refractory period. Ten Langendorff-perfused ferret hearts were instrumented to measure isovolumic left ventricular pressure (LVP) and the aequorin luminescence. Biphasic square-wave electric currents (±20 mA, total duration 30 ms) were delivered between pairs of electrodes. Six hearts were perfused at different extracellular Ca2+ concentrations ([Ca2+]o; 1, 2, 4, and 8 mM). These signals increased LVP from 50.0 ± 9.4 to 70.1 ± 14.7, from 67.5 ± 11.0 to 79.0 ± 15.6, from 79.3 ± 21.0 to 87.1 ± 22.8, and from 84.6 ± 24.0 to 91.8 ± 28.5 mmHg at the respective [Ca2+]o ( P< 0.05). Peak free intracellular [Ca2+] ([Ca2+]i) increased from 0.52 ± 0.13 to 1.37 ± 0.23, from 0.76 ± 0.23 to 1.73 ± 0.14, from 1.10 ± 0.24 to 2.05 ± 0.33, and from 1.41 ± 0.36 to 2.24 ± 0.36 μM/ml, respectively ( P < 0.001). With the use of 1 mg/l propranolol with 1 mM [Ca2+]o, LVP and [Ca2+]i were increased significantly from 48.7 ± 8.18 to 56.3 ± 6.11 mmHg and from 0.61 ± 0.11 to 1.17 ± 0.20 μM, respectively ( P < 0.05). In conclusion, positive inotropism of such electrical currents was due to increased peak [Ca2+]i and Ca2+responsiveness of the myofilaments did not change significantly.

Ca2+ buffering in the heart: Ca2+ binding to and activation of cardiac myofibrils

The measurement of cardiac Ca2+ transients using spectroscopic Ca2+ indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Ca2+ buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Ca2+ dissociation rate constants and which would account for the observed effects of 19F-NMR indicators on the cardiac Ca2+ transient in the Langendorff-perfused ferret heart. It is generally assumed that the Ca2+ dependency of myofibril activation in cardiac myocytes is mediated by a single Ca2+-binding site on troponin C. A model based on 1:1 Ca2+ binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Ca2+ binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Ca2+ binding, giving 58 and 2.0 μM respectively. In addition to reproducing our experimental data on the concentration of free Ca2+ ions in the cytoplasm ([Ca2+]i), the resulting Ca2+ and ATP affinities given by fitting of the model also provided good predictions of the Ca2+ dependence of the myofibrillar ATPase activity measured under in vitro conditions. Solutions to the model also indicate that the Ca2+ mobilized during each beat remains unchanged in the presence of the additional buffering load from Ca2+ indicators. The new model was used to estimate the extent of perturbation of the Ca2+ transient caused by different concentrations of indicators. As little as 10 μM of a Ca2+ indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Ca2+]i and 30 μM will cause approx. 50% reduction in the peak-systolic [Ca2+]i in a heart paced at 1.0 Hz.

Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart

Spectroscopic Ca2+-indicators are thought to report values of free intracellular Ca2+ concentration ([Ca2+]i) that may differ from unperturbed values because they add to the buffering capacity of the tissue. To check this for the heart we have synthesized a new 19F-labelled NMR Ca2+ indicator, 1,2-bis-[2-bis(carboxymethyl)amino-4,5-difluorophenoxy]ethane (‘4,5FBAPTA’), with a low affinity (Kd 2950 nM). The new indicator and four previously described 19F-NMR Ca2+ indicators 1,2-bis-[2-(1 - carboxyethyl)(carboxymethyl)amino - 5 - fluorophenoxy]ethane (‘DiMe-5FBAPTA’), 1,2-bis-[2-(1-carboxyethyl)(carboxymethyl)amino-4-fluorophenoxy]ethane (‘DiMe-4FBAPTA’), 1,2-bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane (‘5FBAPTA’) and 1,2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane (‘MFBAPTA’), with dissociation constants for Ca2+ ranging from 46 to 537 nM, have been used to measure [Ca2+]i, over the range from less than 100 nM to more than 3 μM, in Langendorff-perfused ferret hearts (30 °C, pH 7.4, paced at 1.0 Hz) by 19F-NMR spectroscopy. Loading hearts with indicators resulted in buffering of the Ca2+ transient. The measured end-diastolic and peak-systolic [Ca2+]i were both positively correlated with indicator Kd. The positive correlations between indicator Kd and the measured end-diastolic and peak-systolic [Ca2+]i were used to estimate the unperturbed end-diastolic and peak-systolic [Ca2+]i by extrapolation to Kd = 0 (diastolic) and to Kd = ∞ (systolic) respectively. The extrapolated values in the intact beating heart were 161 nM for end-diastolic [Ca2+]i and 2650 nM for peak-systolic [Ca2+]i, which agree well with values determined from single cells and muscle strips.

Changes in ventricular repolarization during acidosis and low-flow ischemia

Myocardial ischemia, primarily a metabolic insult, is also defined by altered cardiac mechanical and electrical activity. We have investigated the metabolic contributions to the electrophysiological changes during low-flow ischemia (7.5% of the control flow) using31P NMR spectroscopy to monitor metabolic parameters, suction electrodes to study epicardial monophasic action potentials, and 86Rb as a tracer for K+-equivalent efflux during low-flow ischemia in the Langendorff-perfused ferret heart. Shortening of the action potential duration at 90% repolarization (APD90) was most marked between 1 and 5 min after induction of ischemia, at which time it shortened from 261 ± 4 to 213 ± 8 ms. The period of marked APD90 shortening was accompanied by a fivefold increase in the rate of86Rb efflux, both of which were inhibited by the ATP-sensitive K+(KATP)-channel blockers glibenclamide and 5-hydroxydecanoate (5-HD), as well as by a significant fall in intracellular pH (pHi) from 7.14 ± 0.02 to 6.83 ± 0.03 but no change in intracellular ATP concentration ([ATP]i). We therefore investigated whether a fall in pHi could be the metabolic change responsible for modulating cardiac KATP channel activity in the intact heart during ischemia. Both metabolic (30 mM lactate added to extracellular solution) and respiratory ([Formula: see text] increased to 15%) acidosis caused an initial lengthening of APD90 to 112 ± 1.5 and 113 ± 0.9%, respectively, followed by shortening during continued acidosis to 106 ± 1.2 and 106 ± 1.4%, respectively. The shortening of APD90 during continued acidosis was inhibited by glibenclamide, consistent with acidosis causing activation of KATP channels at normal [ATP]i. The similar responses to metabolic (induced by adding either l- or d-lactate) and respiratory acidosis suggest that lactate has no independent metabolic effect on action potential repolarization.