Technical Reference #217

217.

Slow intercellular Ca2+ signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes Sylvia O. Suadicani; Monique J. Vink; David C. Spray, Albert Einstein College of Medicine, American Journal of Physiology: Heart and Circulatory Physiology, 279(217), (2000)
Link To Paper

Abstract:
Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca21 waves that propagated with mean velocities of ;14 mm/s reaching ;80% of the cells in the field. Deletion of connexin43 (Cx43) the main cardiac g

Keywords:
calcium waves; connexin; gap junctions; purinergic receptors; intercellular communications

Materials & Methods:
Dissociation of Neonatal Hearts and Preparation of Primary Cultures of Cardiac Myocytes Wild-type cultures. Neonatal mice (C57BL/6NCr1BR Charles River Laboratories Wilmington MA) were killed by decapitation and the hearts were isolated and placed in 60-mm plastic culture dishes containing sterile ice-cold Dulbecco’s phosphate-buffered saline (PBS; GIBCO-BRL Grand Island NY). After rinsing with PBS to remove the blood we throughly minced the hearts in the dissociation solution [containing 1.25% pancreatin (GIBCO-BRL) and 300 mg of bovine serum albumin (BSA; Sigma St. Louis MO) diluted in (in g/100 ml) 8.0 NaCl 0.2 KCl 0.05 Na2HPO4 1.0 NaHCO3 and 2.0 dextrose; pH 7.1–7.2]. The homogenate was then transferred to a 25-ml Ehrlenmeyer flask with 7 ml of the dissociation solution and placed in a water bath (37°C) for 10 min under continuous stirring. The supernatant was collected in a conical 15-ml tube and spun at 1500 g for 4 min and the pellet was ressuspended in 3 ml of Dulbecco’s modified Eagle’s medium (DMEM) [containing 10% fetal bovine serum (GIBCO-BRL) and 1% penicillin/streptomycin (GIBCO-BRL)]. The tube with the dissociated cells was then placed in the incubator (36–37°C 5% CO2). This procedure was repeated five to seven times or until the heart tissue was totally dissociated. The cells were pooled and preplated in 100-mm plastic culture dishes to which fibroblasts adhered for 1 h. The nonadhered cells were plated onto confocal imaging dishes (Glass Bottom Microwells Uncoated Dishes MatTek) placed in the incubator and allowed to settle for 24 h. After this period we washed the dishes with DMEM to remove the nonadherent cells and fed the cells with 2 ml of DMEM supplemented with cytosine b-D-arabino-furanoside (12.2 mg/50 ml media; Sigma) to inhibit fibroblast growth. Cx43-null cultures. In the case of Cx43-null mice which die shortly after birth (42) the litters bred from Cx43 heterozygous mice (C57BL/6J-Gja1tm1Kdr Jackson Laboratories) were used immediately after birth with the same procedure used for the wild-type animals except that the cardiac myocyte cultures were prepared individually from each of the siblings which were subsequently genotyped from tail DNA as described previously (16). Calcium Imaging and Data Analysis The experiments were performed within 3–5 days after plating the cells. The neonatal mouse heart cells plated on confocal imaging dishes were incubated for 45 min at 37°C with 10 mM of the ratiometric Ca21 indicator indo 1-acetoxymethyl ester (indo 1-AM; Molecular Probes Eugene OR) and rinsed three times with DMEM which was replaced by Tyrode solution [(in mM) 137.0 NaCl 2.7 KCl 0.5 MgCl2 1.8 CaCl2 12.0 NaHCO3 0.5 NaH2PO4 5.5 glucose and 5 HEPES; pH 7.1–7.2] shortly before the experiments. The experiments were conducted at room temperature (21°C) and in some cases (as specified) also at 34°C. The ratio of indo 1-AM fluorescence intensity emitted at two wavelengths (390–440 nm and .440 nm) was imaged using ultraviolet laser excitation at 351 nm. Ratio images were continuously acquired at 1 Hz after background and shading correction using a Nikon real-time confocal microscope (RCM 8000) with a large ultraviolet pinhole and a Nikon 340 water immersion objective (numerical aperture 1.15; working distance 0.2 mm). The intercellular Ca21 waves were induced by focal mechanical stimulation of one myocyte with a glass pipette (outer diameter 1–2 mm). Ratiometric images were saved on the optical disk recorder (OMDR) as the average of 32 frames. The images were further analyzed for measurements of changes in calcium level during playback using Polygon-Star software (Nikon) which averages the gray levels (number of pixels/area) within the regions of interest (circular spots placed on each cell) as a function of elapsed time. The data generated by this software were plotted in graphs as the indo 1-AM fluorescence ratio values versus time (in s) using Microsoft ORIGIN software. The phenomenon of intercellular Ca21 signaling was analyzed in terms of velocity amplitude and efficacy of the Ca21 wave propagation. The velocities of Ca21 wave propagation between cardiac myocytes were calculated as the distance (in mm) between the stimulated and all nonstimulated cells present in the confocal field divided by the time interval (in s) between the half-maximal calcium increases within the stimulated and responding cells. The distances between one stimulated cell and another cell in the field were calculated from the micrographs of the fields (see Fig. 2) as the length of a straight line connecting the centers of the regions of interest. Half-maximal calcium increases were obtained from sigmoidal curves fitted to the ascending phases of the indo 1-AM fluorescence ratio increases using ORIGIN software (Fig. 1). Amplitudes of Ca21 waves were considered to be the maximal increments in intracellular calcium observed in responding cells calculated for each cell as the value of the indo 1-AM fluorescence ratio rise at the peak of the response divided by the basal fluorescence ratio value acquired before the induction of the calcium waves. The efficacy of the Ca21 signal communication is reported here as the number of cells responding with a detectable increase in intracellular calcium [.10% over basal; calibration with Ca21 standards (see Ref. 48) indicates that this corresponds to an approximate doubling of intracellular Ca21 concentration] during the propagation of the wave in relation to the total number of cells within the field. To compare the combined contribution of these three parameters in the communication of the Ca21 signal under different conditions we used the EVA factor defined as the product of the relative values (experimental/ control) obtained for the efficacy velocity and amplitude of the calcium waves (see Ref. 48).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
Cardiac myocytes