Cytosolic free calcium concentration in individual cardiac myocytes in primary culture

Abstract

Cytosolic free Ca2+ concentration, [Ca2+]i, of single isolated Ca2+-tolerant rat ventricular myocytes in primary culture was determined by digital video imaging of intracellular fura-2 fluorescence. In deenergized myocytes in which contractile elements were uncoupled by 2,3-butanedione monoxime, the maximum and minimum fluorescence intensity ratio values of fura-2 in the cell were similar when compared with those of fura-2 solutions observed in the microscope. Through the use of in vitro calibration, [Ca2+]i in quiescent, rod-shaped myocytes was 90 +/- 6 nM. There was no detectable spatial heterogeneity in [Ca2+]i in resting myocytes. Localized regions of [Ca2+]i elevation were observed in cells undergoing spontaneous rhythmic contractions or when subjected to mild depolarization by KCl. Additions of gramicidin or veratridine resulted in massive increases in [Ca2+]i (greater than 1 microM) and immediate cell hypercontracture. Ruthenium red elicited a modest increase in [Ca2+]i but extracellular ATP or epinephrine had no effect. We conclude the following: 1) digital video imaging of resting cardiac cells did not reveal any subcellular Ca2+ gradients; 2) the fluorescence properties of intracellular fura-2 were similar to that in free solution; and 3) subcellular heterogeneity of [Ca2+]i in isolated myocytes was observed in cells undergoing spontaneous rhythmic contraction.

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes

“…Background and cellular autofluorescence measured in sham-GFP and MI-GFP myocytes not loaded with fura 2 accounted for Ͻ10% of the fura 2 signal (34). Intracellular fura 2 fluorescence was calibrated daily for each batch of myocytes, as previously described (7). [Ca 2ϩ ]i transient data derived from fura 2 signals were analyzed with custom-written software (Ionoptix) (22,25,28,29,(32)(33)(34).…”

H2O2-induced Ca2+overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

“…In this equation, the R value is the ratio of fluorescence with excitation at 340 and 380 nm, and ␤ is the ratio of fluorescence with excitation at 380 nm in 0 Ca 2ϩ to that saturating Ca 2ϩ . Kd is the effective dissociation constant for fura 2 and was used as 224 nM (4,7,20). The data were analyzed with Merlin software and graphed using Delta Graph version 4.5 software for Macintosh.…”

Endothelin and angiotensin II stimulation of Na+-H+ exchange is impaired in cardiac hypertrophy.

“…Two to four experiments were performed in sequence from separate coverslips of myocytes isolated from one heart. We have documented recently the stability of both cell motion and [Ca ] i under baseline conditions in hypertrophied and control myocytes, we performed calibration studies described (3) by a modification of the methods of Cheung et al (25) and Borzak et al (26). The calibration methods have been described in detail elsewhere (3).…”