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

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.

High [Ca2+]o-induced electrical heterogeneity and extrasystolic activity in isolated canine ventricular epicardium. Phase 2 reentry.

“…unpublished observation). Elevated intracellular calcium activity, known to occur with high [Ca2+] and rapid stimulation,22 24 can lead to the development of DADs and DAD-induced triggered activity in the ventricular conducting system18 as well as to deterioration of intracellular communication and major slowing of conduction.2021 Neither was observed under the relatively mild conditions used in the present study. It is noteworthy that canine ventricular epicardium and endocardium have previously been shown to be resistant to agents that induce DADs, including high [Ca2+]0, digitalis, and the calcium agonist Bay K 8644.19,40,41 These agents readily induce DADs and triggered activity in tissues from the M region of the canine ventricle and Purkinje system but not in those isolated from the epicardial and endocardial surfaces.…”

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

“…Hearts were allowed to equilibrate for 40 min following loading of indicators, before the acquisition of data. The concentration of indicator in the cytosolic space is proportional to the area of the "*F-NMR resonances of the indicator and, in a previous paper, using $H-labelled 5FBAPTA, we showed that, at a S\N ratio of 10 : 1, the intracellular concentration of 5FBAPTA was 120 µM [21]. All indicators were loaded to similar S\N ratio levels and it was assumed that the final cytosolic concentration of each of the indicators reached approx.…”

“…The magnet was shimmed using the perfusate proton signal : "H line widths at a half-height of less than 60 Hz were routinely obtained. End-diastolic [Ca# + ] i and the Ca# + transients were obtained using gated pulse sequences as described previously [8,21].…”

The effect of Mg2+ on cardiac muscle function: is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

“…Upper panel : isolated ferret heart perfused with 5FBAPTA at 30 mC, with data from [69]. Lower panel : isolated ferret papillary muscle loaded with aequorin at 30 mC, with data taken from [70].…”

“…In our own work the clearest indication of the complexity of Ca# + activation, as described above, is found when comparing under different conditions the relationship of the time course of the Ca# + transient with that of the associated contraction. Figure 5 shows this comparison in the presence and absence of added Ca# + buffering, the heavily buffered heart, using 5FBAPTA, taken from Harding et al [69] (upper panel), and the very weakly buffered aequorin-loaded heart muscle, taken from Yue et al [70] (lower panel), both plotted on the same time scale. We do not show the data acquired before the loading of 5FBAPTA, when the contraction resembles that for aequorin with a delay of only a few milliseconds, due to the mechanics of the measurement procedure.…”