1. Ventricular myocytes isolated from ferret or cat were loaded with the acetoxymethyl ester form of indo-1 (indo-1 AM) such that ~75% of cellular indo-1 was mitochondrial. The intramitochondrial indo-1 concentration was 0.5-2 mM. 2. Myocytes were also voltage clamped (membrane capacitance, C(m) = 100 pF) and a typical wash-out time constant of cytosolic indo-1 by a patch pipette was found to be ~300 s. Depolarizations to +110 mV produced graded and progressive cellular Ca2+ load via Na+-Caaa2+ exchange. 3. During these relatively slow Ca2+ transients, cell contraction (ΔL) paralleled fluorescence ratio signals (R) such that ΔL could be used as a bioassay of cytosolic [Ca2+] ([Ca2+](c)), where [Ca2+](CL) is the inferred signal which is delayed by ~200 ms from true [Ca2+](c). 4. In myocytes without Mn2+ quench, the kinetics of the total cellular indo-1 signal, ΔR (including cytosolic and mitochondrial components), match ΔL during stimulations at low basal [Ca2+](i). However, after progressive Ca2+ loading, ΔR kinetics deviate from ΔL dramatically. The deviation can be completely blocked by a potent mitochondrial Ca2+ uniport blocker, Ru360. 5. When cytosolic indo-1 is quenched by Mn2+, initial moderate stimulation triggers contractions (ΔL), but no change in indo-1 signal, indicating both the absence of cytosolic Ca2+-sensitive indo-1 and unchanged mitochondrial [Ca2+] (Δ[Ca2+](m)). Subsequent stronger stimulation evoked larger ΔL and also ΔR. The threshold [Ca2+](c) for mitochondrial uptake was 300-500 nM, similar to that without Mn2+ quench. 6. At high Ca2+ loads where Δ[Ca2+](m) is detected, the time course of [Ca2+](m) was different from that of [Ca2+](c). Peak [Ca2+](m) after stimulation has an ~1 s latency with respect to [Ca2+](c), and [Ca2+](m) decline is extremely slow. 7. Upon a Ca2+ influx which increased [Ca2+](c) by 0.4 μM and [Ca2+](m) by 0.2 μM, total mitochondrial Ca2+ uptake was ~13 μmol (1 mitochondria)-1. 8. With Mn2+ quench of cytosolic indo-1, there was no mitochondrial uptake of Mn2+ until the point at which mitochondrial Ca2+ uptake became apparent. However, after mitochondrial Ca2+ uptake starts, mitochondria continually take up Mn2+ even during relaxation, when [Ca2+](c) is low. 9. It is concluded that mitochondria in intact myocytes do not take up detectable amounts of Ca2+ during individual contractions, unless resting [Ca2+](c) exceeds 300-500 nM. At high cell Ca2+ loads and [Ca2+](c), mitochondrial Ca2+ transients occur during the twitch, but with much slower kinetics than those of [Ca2+](c).
ASJC Scopus subject areas