Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: II. Ventricular myocytes

Takashi Miyahara; Robert E. Akins; Rocky S. Tuan

摘要

AbstractWe have previously shown that cardiovascular anomalies, such as hypertension and tachycardia, develop in Ca2+‐deficient, shell‐less (SL) chick embryos cultured ex ovo, accompanied by elevated circulating catecholamines and higher α‐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 Ca2+‐free buffer and then analyzed by ratiometric fluorescence (350 nm/380 nm) microscopy for kinetic changes in Ca2+i(R350/380values) as a function of Ca2+oand 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+icompared to NL cells. At higher Ca2+o(3–5 mM), there was no significant difference in Ca2+iand beating rate between NL and SL cells. Treatment with norepinephrine (NE; 0.01–1 μM) at 1 mM Ca2+osubstantially increased Ca2+iin both NL and SL cells. In the former, the NE effect was completely inhibited by β‐blockade (1 μM propranolol). In contrast, in SL cells, NE remained effective after β‐blockade, and combined α‐blockade (1 μM prazosin) and β‐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 C

Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: II. Ventricular myocytes

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