DISTRIBUTION OF ELECTRICAL POTENTIAL, PH, FREE CA(2)-RABBIT CARDIAC MYOCYTES DURING CHEMICAL HYPOXIA - A MULTIPARAMETER DIGITIZED CONFOCAL MICROSCOPIC STUDY(, AND VOLUME INSIDE CULTURED ADULT)

Exploiting the optical sectioning capabilities of laser scanning confo cal microscopy and using parameter-specific fluorescent probes, we det ermined the distribution of pH, free Ca2+, electrical potential, and v olume inside cultured adult rabbit cardiac myocytes during ATP depleti on and reductive stress with cyanide and 2-deoxyglucose (''chemical hy poxia''). During normoxic incubations, myocytes exhibited a cytosolic pH of 7.1 and a mitochondrial Ph of 8.0 (Delta pH = 0.9 units). Sarcol emmal membrane potential (Delta Psi) was -80 mV, and mitochondrial Del ta Psi was as high as -100 mV, yielding a mitochondrial protonmotive f orce (Delta rho) of -155 mV (Delta P = Delta Psi - 60 Delta pH). After 30 min of chemical hypoxia, mitochondrial Delta pH decreased to 0.5 p H units, but mitochondrial Delta Psi remained essentially unchanged. B y 40 min, Delta pH was collapsed, and mitochondrial and cytosolic free Ca2+ began to increase. Mitochondrial and sarcolemmal Delta Psi remai ned high. As Ca2+ rose, myocytes shortened, hypercontracted, and blebb ed with a 30% decrease of cell volume. After hypercontraction, extensi ve mitochondrial Ca2+ loading occurred. After another few minutes, mit ochondria depolarized completely and released their load of Ca2+. Afte r many more minutes, the sarcolemmal permeability barrier broke down, and viability was lost. These studies demonstrate a sequence of subcel lular ionic and electrical changes that may underlie the progression t o irreversible hypoxic injury.