Quantification of [Ca2+]i in perfused hearts. Critical evaluation of the 5F-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion.
Calcium has been implicated as a mediator of cell injury in ischemia and reperfusion, but direct measurements of Ca2+ are required to refine this idea. We used nuclear magnetic resonance spectroscopy and the Ca2+ indicator 5F-BAPTA to measure [Ca2+]i in perfused ferret hearts. Several lines of evidence are presented to show that loading with the acetoxymethyl ester of 5F-BAPTA is not significantly complicated by accumulation of partially de-esterified metabolites, compartmentalization into mitochondria, or disproportionate uptake into endothelial cells. During 20 minutes of total global ischemia at 30 degrees C, time-averaged [Ca2+]i increased significantly, reaching peak values roughly three times control at 15-20 minutes. Reperfusion resulted in a persistent elevation of [Ca2+]i during the first 5 minutes, but not afterward. Although the nonlinear response of 5F-BAPTA to [Ca2+] leads to underestimation of the true time-averaged [Ca2+]i, the measured alterations of intracellular Ca2+ homeostasis during ischemia are large compared with the likely errors in quantification. Phosphorus nuclear magnetic resonance spectroscopy of 5F-BAPTA-loaded hearts reveals changes during ischemia similar to those recorded previously in hearts not containing a Ca2+ indicator. Developed pressure recovers to only 50% of control values during reflow, indicating that the presence of 5F-BAPTA in the cytosol does not protect against stunning, at least when the extracellular calcium concentration has been raised to 8 mM. We conclude that 5F-BAPTA provides useful measurements that reveal that time-averaged [Ca2+]i rises during ischemia and returns to control levels soon after reperfusion.
Marban
E
E
Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Kitakaze
M
M
Koretsune
Y
Y
Yue
D T
DT
Chacko
V P
VP
Pike
M M
MM
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003327:Coronary Disease / Q000378:metabolism* / Q000503:physiopathology; D004533:Egtazic Acid / Q000031:analogs & derivatives; D005289:Ferrets; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D009200:Myocardial Contraction; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000378:metabolism*; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism; D013696:Temperature
D010710:Phosphates; D004533:Egtazic Acid; C039877:5,5'-difluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium