Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: II. Ventricular myocytes.
We have previously shown that cardiovascular anomalies, such as hypertension and tachycardia, develop in Ca(2+)-deficient, shell-less (SL) chick embryos cultured ex ovo, accompanied by elevated circulating catecholamines and higher alpha-adrenergic sensitivity of cardiovascular functions. Results described in the preceding work, using erythrocytes as an experimental system, show that cellular Ca2+ handling properties are also altered as a result of long-term calcium deficiency. To examine the relevance of these findings to cells of the cardiovasculature, we have analyzed and compared the Ca2+ handling characteristics of the heart cells of SL and normal (NL) embryos. For this study, isolated and cultured ventricular myocytes of SL and NL embryos were loaded with Fura-2 via transient membrane damage with glass beads. Compared to Fura-2/AM, bead loading yielded similar values and kinetic profiles of [Ca2+]i-dependent differential fluorescence and, in addition, did not affect cell viability and beating activity. The Fura-2 loaded ventricular myocytes were washed in Ca(2+)-free buffer and then analyzed by ratiometric fluorescence (350 nm/380 nm) microscopy for kinetic changes in [Ca2+]i (R350/380 values) as a function of [Ca2+]o and adrenergic modifiers. At 0.5 and 1.0 mM [Ca2+]o, SL cells showed significantly higher [Ca2+]i, higher beating rates, and faster rate of increase in [Ca2+]i compared to NL cells. At higher [Ca2+]o (3.5 mM), there was no significant difference in [Ca2+]i and beating rate between NL and SL cells. Treatment with norepinephrine (NE; 0.01-1 microM) at 1 mM [Ca2+]o substantially increased [Ca2+]i in both NL and SL cells. In the former, the NE effect was completely inhibited by beta-blockade (1 microM propranolol). In contrast, in SL cells, NE remained effective after beta-blockade, and combined alpha-blockade (1 microM prazosin) and beta-blockade was needed to inhibit completely the NE effect. In both NL and SL cells, treatment with NE substantially increased beating rates in a similar manner. Taken together, these findings suggest that Ca2+ handling and adrenergic regulation of the heart cells are significantly altered in the SL embryos, and that these alterations may be related to the development of impaired cardiovascular functions resulting from systemic Ca2+ deficiency.