CYTOSOLIC AND MITOCHONDRIAL CA2+ SIGNALS IN PATCH CLAMPED MAMMALIAN VENTRICULAR MYOCYTES

1. Ventricular myocytes isolated from ferret or cat were loaded with t he acetoxymethyl ester form of indo-1 (indo-1. AM) such that similar t o 75% of cellular indo-1 was mitochondrial. The intramitochondrial ind o-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 const ant of cytosolic indo-1 by a patch pipette was found to be similar to 300 s. Depolarizations to +110 mV produced graded and progressive cell ular Ca2+ load via Na+-Ca2+ exchange. 3. During these relatively slow Ca2+ transients, cell contraction (Delta L) paralleled fluorescence ra tio signals (R) such that Delta L could be used as a bioassay of cytos olic [Ca2+] ([Ca2+](c)), where [Ca2+](CL) is the inferred signal which is delayed by similar to 200 ms from true [Ca2+](c). 4. In myocytes w ithout Mn2+ quench, the kinetics of the total cellular indo-1 signal, Delta R (including cytosolic and mitochondrial components), match Delt a L during stimulations at low basal [Ca2+](i). However, after progres sive Ca2+ loading, Delta R kinetics deviate from Delta L dramatically. The deviation carl be completely blocked by a potent mitochondrial Ca 2+ uniport blocker, Ru360. 5. When cytosolic indo-1 is quenched by Mn2 +, initial moderate stimulation triggers contractions (Delta L), but n o change in indo-1 signal, indicating both the absence of cytosolic Ca 2+-sensitive indo-1 and unchanged mitochondrial [Ca2+] (Delta[Ca2+](m) ). Subsequent stronger stimulation evoked larger Delta L and also Delt a R. The threshold [Ca2+](c) for mitochondrial Ca2+ uptake was 300-500 nM, similar to that without Mn2+ quench. 6. At high Ca2+ loads where Delta[Ca2+], is detected, the time course of [Ca2+](m) was different f rom that of [Ca2+](c). Peak [Ca2+](m) after stimulation has an similar to 1 s latency with respect to [Ca2+](c), and [Ca2+](m) decline is ex tremely slow. 7. Upon a Ca2+ influx which increased [Ca2+](c) by 0.4 m u M and [Ca2+](m) by 0.2 mu M, total mitochondrial Ca2+ uptake was sim ilar to 13 mu mol (1 mitochondria)(-1). 8. With Mn2+ quench of cytosol ic indo-1, there was no mitochondrial uptake of Mn2+ until the point a t which mitochondrial Ca2+ uptake became apparent. However, after mito chondrial Ca2+ uptake starts, mitochondria continually take up Mn2+ ev en during relaxation, when [Ca2+](c) is low. 9. It is concluded that m itochondria in intact myocytes do not take up detectable amounts of Ca 2+ during individual contractions, unless resting [Ca2+](c) exceeds 30 0-500 nM. At high cell Ca2+ loads and [Ca2+](c), mitochondrial Ca2+ tr ansients occur during the twitch, but with much slower kinetics than t hose of [Ca2+](c).