Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes

• Published in: The Journal of physiology 1998-03, Vol.507 (2), p.379-403
• Main Authors: Zhou, Zhuan, Matlib, Mohammed A., Bers, Donald M.
• Format: Article
• Language: English
• Subjects: Animals; Calcium Channels - drug effects; Calcium Channels - metabolism; Cats; Cells, Cultured; Cytosol - metabolism; Electrophysiology; Ferrets; Fluorescent Dyes; Heart Ventricles - drug effects; Heart Ventricles - metabolism; Heart Ventricles - ultrastructure; Kinetics; Manganese - pharmacology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Microscopy, Fluorescence; Mitochondria, Heart - drug effects; Mitochondria, Heart - metabolism; Myocardium - metabolism; Myocardium - ultrastructure; Original; Patch-Clamp Techniques
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.1998.379bt.x

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 m m . 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 Ca 2+ load via Na + -Ca 2+ exchange. During these relatively slow Ca 2+ transients, cell contraction (Δ L ) paralleled fluorescence ratio signals ( R ) such that Δ L could be used as a bioassay of cytosolic [Ca 2+ ] ([Ca 2+ ] c ), where [Ca 2+ ] CL is the inferred signal which is delayed by ∼200 ms from true [Ca 2+ ] c . In myocytes without Mn 2+ quench, the kinetics of the total cellular indo-1 signal, Δ R (including cytosolic and mitochondrial components), match Δ L during stimulations at low basal [Ca 2+ ] i . However, after progressive Ca 2+ loading, Δ R kinetics deviate from Δ L dramatically. The deviation can be completely blocked by a potent mitochondrial Ca 2+ uniport blocker, Ru360. When cytosolic indo-1 is quenched by Mn 2+ , initial moderate stimulation triggers contractions (Δ L ), but no change in indo-1 signal, indicating both the absence of cytosolic Ca 2+ -sensitive indo-1 and unchanged mitochondrial [Ca 2+ ] (Δ[Ca 2+ ] m ). Subsequent stronger stimulation evoked larger Δ L and also Δ R. The threshold [Ca 2+ ] c for mitochondrial Ca 2+ uptake was 300–500 nM, similar to that without Mn 2+ quench. At high Ca 2+ loads where Δ[Ca 2+ ] m is detected, the time course of [Ca 2+ ] m was different from that of [Ca 2+ ] c . Peak [Ca 2+ ] m after stimulation has an ∼1 s latency with respect to [Ca 2+ ] c , and [Ca 2+ ] m decline is extremely slow. Upon a Ca 2+ influx which increased [Ca 2+ ] c by 0.4 μ m and [Ca 2+ ] m by 0.2 μ m , total mitochondrial Ca 2+ uptake was ∼13 μmol (l mitochondria) −1 . With Mn 2+ quench of cytosolic indo-1, there was no mitochondrial uptake of Mn 2+ until the point at which mitochondrial Ca 2+ uptake became apparent. However, after mitochondrial Ca 2+ uptake starts, mitochondria continually take up Mn 2+ even during relaxation, when [Ca 2+ ] c is low. It is concluded that mitochondria in intact myocytes do not take up detectable amounts of Ca 2+ during individual contractions, unless resting [Ca 2+ ] c exceeds 300–500 nM. At hig