Cytosolic calcium transients from the beating mammalian heart.

Abstract

To elucidate the role of cytosolic calcium, [Ca2+]1, in the physiology of the normal and ischemic heart, we have developed a method for recording [Ca2+]1 transients from the epicardial surface of the rabbit ventricle after arterial perfusion with the cell-permeant cytosolic calcium indicator indo-l AM. Hearts were illuminated at 360 nm, and fluorescence was recorded simultaneously at 400 and 550 nm. The F4N/F550 fluorescence ratio was calculated by an analog circuit that allowed cancelation of small movement artifacts that were present at single wavelengths. Clear [Ca2+] METHODSAlbino male New Zealand rabbits weighing between 1.8 and 2.2 kg were sacrificed by cervical fracture. The heart was rapidly excised and perfused with a saline solution at a constant aortic flow rate of 20-30 ml/min. The perfusate contained 115 mM NaCl, 4.7 mM KCI, 2.0 mM CaC12, 0.7 mM MgCl2, 28 mM NaHCO3, 0.5 mM NaH2PO4, 20 mM glucose, 10 units of insulin per liter, and 0.1% fetal calf serum adjusted to pH 7.4, equilibrated with 95% 02/5% C02, and heated to maintain epicardial temperature at 30'C + 1PC. Contractions of the left ventricle were measured with an intracavitary latex balloon that contained a fiber-optic pressure transducer (Camino Laboratories) or with an epicardial strain gauge that had the advantage of monitoring wall stress near the site at which fluorescence was measured. The left ventricle was vented by a small polyethylene catheter that drained thebesian vein flow. Some hearts were paced at 180/min by an epicardial plunge electrode.Fluorescence excitation was provided by a 100-W Hg vapor lamp. Illumination was filtered at 360 ± 5 nm and directed through a silica fiber-optic cable onto a circular region of epicardium 1 cm in diameter. Movement artifact was minimized by attachment of the fiber-optic cables to the heart using a plastic hub and rubber girdle. Fluorescence was collected by a ring of eight coaxial fiber-optic cables, divided by a beam splitter, and then filtered at 400 ± 12.5 nm and 550 ± 20 nm before reaching photomultiplier tubes (Hamamatsu). The above emission wavelengths were found to give clearer fluorescence transients than the in vitro fluorescence maxima of indo-1 (Ca2' bound = 405 nm; Ca2+ free = 480 nm). Our chosen emission wavelengths allow greater rejection of the noncalcium-dependent fluorescence of unhydrolyzed indo-1 AM (fluorescence maximum = 450 nm), which is retained in tissue after loading (9). Photomultiplier output at the two emission wavelengths was entered into an electronic ratio circuit and the fluorescence ratio, F4w/F550, was displayed on a strip-chart recorder.Indo-1 AM was solubilized in a mixture of dimethyl sulfoxide/pluronic F-127 (25%, wt/vol), 1 ml of which was added to 400 ml of Tyrodes solution containing 5% fetal calf serum. The final concentration of indo-1 AM was 2.5 AM.Perfusion with indo-1 AM continued for 30 min; this was followed by a 30-min washout. This method of indicator loading produced a 5-to 12-fold increase in fluorescence at Abbreviation: [Ca21],, ...

Continuous fluorimetric assessment of the changes in cytoplasmic calcium concentration during exposure of rat isolated myocardium to conditions of simulated ischaemia

“…Previous workers have obtained similar findings with both intact ventricular myocardium and isolated ventricular myocytes, and have concluded that this optical phenomenon represents the systolic rise and diastolic fall in [Ca], upon which contraction and relaxation depend (Dubell & Houser, 1987a; Lee et al, 1987;1988). Both the systolic rise in the F400 signal and simultaneous fall in the F500 signal also reflect the rise in [Ca],.…”

“…Both the systolic rise in the F400 signal and simultaneous fall in the F500 signal also reflect the rise in [Ca],. However, calculated changes in the ratio of emission intensities at 400 nm and 500 nm respectively were larger than the changes observed in either the F400 or the F500 signal separately, and the ratio has been shown to be less subject to interference and motion artefact (Lee et al, 1987;1988;Eisner et al, 1989). For these reasons the F400/ 500 ratio, rather than its components was used routinely in the present work.…”

“…In that way a rise and a subsequent fall in [Ca], may be demonstrated with each heart beat ; Lee et al, 1987;1988;Stern et al, 1988;Eisner et al, 1989;Kohmoto & Barry, 1989;Morris et al, 1989).…”

“…A raised cytoplasmic concentration ([Ca]c) has been demonstrated during periods of depletion of the myocardial stores of adenosine 5'-triphosphate (ATP), and this occurs during ischaemia, for example ; Lee et al, 1987;1988;Eisner et al, 1989;Kohmoto & Barry, 1989;Morris et al, 1989). These observations have been made with non-toxic fluorescent dyes, such as indo-1, that are sensitive to even the very low levels of [Ca], that normally prevail during diastole.…”

Influence of ischemic preconditioning in myocardial protection in patients undergoing myocardial revascularization with intermittent crossclamping of the aorta. Analysis of ions and blood gases

“…Several authors 19,25,26 suggest that the increase in the intracellular calcium is one of the major determinants of the ischemic lesion, through a calcium-dependent mechanism that activates the catabolic enzymes, leading to irreversible damage of the plasmatic membrane, and determining cellular death.…”