Calcium Depletion and Repletion in Cultured Chick Heart Muscle Cells

Abstract

Calcium-free incubation followed by exposure to calcium damages naturally occurring cardiac muscle preparations irreversibly. Whether the observed calcium overload during calcium repletion is a primary cause for, or a secondary consequence of, sarcolemmal disruption and cell injury is controversial. We used cultured embryonic chicken heart muscle cells to correlate ionic, metabolic, and ultrastructural changes during calcium depletion (0Ca. 1 mM EGTA) and repletion. After 10 min of calcium depletion, intracellular Na increased four-fold above control levels, intracellular K decreased by 26% total cell Ca decreased by 81%, and cytosolic ionized Ca concentration decreased by 87%. Within 10 min of subsequent calcium repletion, total cell Ca transiently increased to four-fold above control, cytosolic ionized Ca concentration transiently increased to twice control, and both Na and K returned toward control levels; by 3 h of calcium repletion, physiological cation (Na, K, Ca) contents were restored and adenine nucleotide contents were normal. Long-term (i.e. 120 min) calcium depletion did not significantly reduce cell ATP levels, but increased adenine nucleotide turnover as indicated by adenosine and lactate release; after 60 min of subsequent calcium repletion, ionic and metabolic parameters were returned to control levels. During calcium depletion (both short- and long-term) and subsequent repletion, no ultrastructural changes occurred. When Mg was also removed during calcium depletion, the ionic changes during depletion and subsequent repletion were enhanced. When 10 μM CCCP was present during calcium depletion and repletion, cytosolic ionized Ca concentration increased to six-fold above control with no increase in total cell Ca content, suggesting that the increased Ca is buffered, in part, by mitochondria. These results indicate that an increase in Ca per se, occurring when high energy phosphate levels and/or cellular Ca buffering capacity are maintained, does not seem to be associated with irreversible cell injury.