Cytosolic and mitochondrial [Ca²⁺] in whole hearts using indo-1 acetoxymethyl ester: Effects of high extracellular Ca²⁺

Assessment of free cytosolic [Ca²⁺] ([Ca²⁺]_c) using the acetoxymethyl ester (AM) form of indo-1 may be compromised by loading of indo-1 into noncytosolic compartments, primarily mitochondria. To determine the fraction of noncytosolic fluorescence in whole hearts loaded with indo-1 AM, Mn²⁺ was used to quench cytosolic fluorescence. Residual (i.e., noncytosolic) fluorescence was subtracted from the total fluorescence before calculating [Ca²⁺]_c. Noncytosolic fluorescence was used to estimate mitochondrial [Ca²⁺]. In hearts paced at 5 Hz (N = 17), noncytosolic fluorescence was 0.61 ± 0.06 and 0.56 ± 0.07 of total fluorescence at λ₃₈₅ and λ₄₅₆, respectively. After taking into account noncytosolic fluorescence, systolic and diastolic [Ca²⁺]_c was 673 ± 72 and 132 ± 9 nM, respectively. noncytosolic [Ca²⁺] was 183 ± 36 nM and increased to 272 ± 12 when extracellular Ca²⁺ was increased from 2 to 6 mM. This increase in noncytosolic [Ca²⁺] was inhibited by ruthenium red, a blocker of Ca²⁺ uptake by mitochondria. We conclude that cytosolic and mitochondrial [Ca²⁺] can be determined in whole hearts loaded with indo-1 AM by using Mn²⁺ to quench cytosolic fluorescence.