Dynamic buffering of mitochondrial Ca2+ during Ca2+ uptake and Na+-induced Ca2+ release

Abstract

In cardiac mitochondria, matrix free Ca2+ ([Ca2+]m) is primarily regulated by Ca2+ uptake and release via the Ca2+ uniporter (CU) and Na+/Ca2+ exchanger (NCE) as well as by Ca2+ buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca2+ buffering affects these dynamics under various Ca2+ uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca2+ buffering on the uptake and release of Ca2+, we monitored Ca2+ dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca2+] ([Ca2+]e) and [Ca2+]m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl2] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca2+] and [Ca2+e]m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca2+-induced changes in mitochondrial bioenergetics. Our [Ca2+]e and [Ca2+]m measurements demonstrate that Ca2+ uptake and release do not show reciprocal Ca2+ dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca2+ buffering system in the matrix compartment. The Na+ - induced Ca2+ release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca2+ uptake in the presence of large amounts of CaCl2, but not by Na+- induced Ca2+ release.

Identity and function of a cardiac mitochondrial small conductance Ca 2+ -activated K + channel splice variant

“…This is apparent from a previous report [33] in which we measured external and matrix [Ca 2+ ] using a similar respiration buffer but containing 0.6 mM CaCl 2 and 1 mM EGTA; this resulted in an external [Ca 2+ ] of 300 nM and a matrix [Ca 2+ ] of 1000 nM. In buffer containing approximately 40 μM EGTA carried over with the mitochondria from the isolation buffer, with no added CaCl 2 , we observed a basal external [Ca 2+ ] of 80–100 nM and a matrix [Ca 2+ ] of 210–250 nM [34,35]. Mitochondrial K + was monitored in a cuvettebased spectrophotometer (QM-8, Photon Technology International, PTI) with excitation and emission light, λ ex 340 and 380 nm; λ em 500 nm, while adding 0–3 mM CaCl 2 to the buffer.…”

Endogenous and Agonist-induced Opening of Mitochondrial Big Versus Small Ca2+-sensitive K+ Channels on Cardiac Cell and Mitochondrial Protection

“…State 3 respiration was determined after adding 250 μM ADP. In other experiments using the same mitochondrial pellet, Ca 2+ retention capacity (CRC) was evaluated by measuring the decline in buffer [Ca 2+ ] with Fura-4 penta-K + using fluorescence spectrophotometry (QM-8, Horiba/Photon Technology International, PTI) as described previously 44–48 during bolus additions of 20 μM CaCl 2 every 90 s. Based on the similar improvement in cardiac function by individual or combined agonists, only the mitochondrial effects of the combined agonists NS1619 and DCEB were examined in the respiration and CRC experiments.…”

Isoflurane modulates cardiac mitochondrial bioenergetics by selectively attenuating respiratory complexes

“…ΔΨ m was measured in the mitochondrial suspension during states 2, 3 and 4 respiration by fluorescence spectrophotometry using the fluorescent dye tetramethyl-rhodamine methyl ester (1 µM TMRM; Molecular Probes, Eugene, OR) at excitation wavelengths 546 nm and 573 nm and emission wavelength 590 nm [31], as we have described [29]. TMRM is a ratiometric, membrane potential sensitive, cationic fluorophore that equilibrates across the inner mitochondrial membrane (IMM) based on its electrochemical potential.…”