Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

Decoding of fast cytosolic Ca²⁺ concentration (Ca²⁺]_i) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial Ca²⁺] (Ca²⁺]_m) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial Ca²⁺] (Ca²⁺]_em) to >0.5 µM resulted in a Ca²⁺]_em-dependent increase in the rate of mitochondrial Ca²⁺ accumulation (Ca²⁺]_em resulting in half-maximal rate of Ca²⁺ accumulation = 4.4 µM) via Ca²⁺ uniporter. Ca²⁺ uptake was sensitive to the Ca²⁺ uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of Ca²⁺]_m increase and recovery were dependent on the extramitochondrial Na⁺] (Na⁺]_em) due to Ca²⁺ extrusion via mitochondrial Na⁺/Ca²⁺ exchanger. The maximal rate of Ca²⁺ extrusion was observed with Na⁺]_em in the range of 20–40 mM. Rapid switching (0.25–1 Hz) of Ca²⁺]_em between 0 and 100 µM simulated rapid beat-to-beat changes in Ca²⁺]_i (with Ca²⁺]_i transient duration of 100–500 ms). No Ca²⁺]_m oscillations were observed, either under conditions of maximal rate of Ca²⁺ uptake (100 µM Ca²⁺]_em, 0 Na⁺]_em) or with maximal rate of Ca²⁺ removal (0 Ca²⁺]_em, 40 mM Na⁺]_em). The slow frequency-dependent increase of Ca²⁺]_m argues against a rapid transmission of Ca²⁺ signals between cytosol and mitochondria on a beat-to-beat basis in the heart. Ca²⁺]_m changes elicited by continuous or pulsatile exposure to elevated Ca²⁺]_em showed no difference in mitochondrial Ca²⁺ uptake. Thus in cardiac myocytes fast Ca²⁺]_i transients are integrated by mitochondrial Ca²⁺ transport systems, resulting in a frequency-dependent net mitochondrial Ca²⁺ accumulation. mitochondrial Ca²⁺; excitation-contraction coupling; cardiomyocytes