scholarly journals Dynamics of matrix-free Ca2+ in cardiac mitochondria: two components of Ca2+ uptake and role of phosphate buffering

2012 ◽  
Vol 139 (6) ◽  
pp. 465-478 ◽  
Author(s):  
An-Chi Wei ◽  
Ting Liu ◽  
Raimond L. Winslow ◽  
Brian O'Rourke

Mitochondrial Ca2+ uptake is thought to provide an important signal to increase energy production to meet demand but, in excess, can also trigger cell death. The mechanisms defining the relationship between total Ca2+ uptake, changes in mitochondrial matrix free Ca2+, and the activation of the mitochondrial permeability transition pore (PTP) are not well understood. We quantitatively measure changes in [Ca2+]out and [Ca2+]mito during Ca2+ uptake in isolated cardiac mitochondria and identify two components of Ca2+ influx. [Ca2+]mito recordings revealed that the first, MCUmode1, required at least 1 µM Ru360 to be completely inhibited, and responded to small Ca2+ additions in the range of 0.1 to 2 µM with rapid and large changes in [Ca2+]mito. The second component, MCUmode2, was blocked by 100 nM Ru360 and was responsible for the bulk of total Ca2+ uptake for large Ca2+ additions in the range of 2 to 10 µM; however, it had little effect on steady-state [Ca2+]mito. MCUmode1 mediates changes in [Ca2+]mito of 10s of μM, even in the presence of 100 nM Ru360, indicating that there is a finite degree of Ca2+ buffering in the matrix associated with this pathway. In contrast, the much higher Ca2+ loads evoked by MCUmode2 activate a secondary dynamic Ca2+ buffering system consistent with calcium-phosphate complex formation. Increasing Pi potentiated [Ca2+]mito increases via MCUmode1 but suppressed [Ca2+]mito changes via MCUmode2. The results suggest that the role of MCUmode1 might be to modulate oxidative phosphorylation in response to intracellular Ca2+ signaling, whereas MCUmode2 and the dynamic high-capacity Ca2+ buffering system constitute a Ca2+ sink function. Interestingly, the trigger for PTP activation is unlikely to be [Ca2+]mito itself but rather a downstream byproduct of total mitochondrial Ca2+ loading.

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Giovanni Fajardo ◽  
Mingming Zhao ◽  
Gerald Berry ◽  
Daria Mochly-Rosen ◽  
Daniel Bernstein

β2-adrenergic receptors (β2-ARs) modulate cardioprotection through crosstalk with multiple pathways. We have previously shown that β2-ARs are cardioprotective during acute exposure to Doxorubicin (DOX). DOX cardiotoxicity is mediated through a Ca 2+ -dependent opening of the mitochondrial permeability transition pore (MPT) and mitochondrial dysfunction, however the upstream signals linking cell surface receptors and the MPT are not clear. The purpose of this study was to assess crosstalk between β2-AR signaling and mitochondrial function in DOX toxicity. DOX 10 mg/kg was administered to β2−/− and WT mice. Whereas there was no mortality in WT, 85% of β2−/− mice died within 30 min (n=20). Pro- and anti-survival kinases were assessed by immunobloting. At baseline, β2−/− showed normal levels of ϵPKC, but a 16% increase in δPKC compared to WT (p<0.05). After DOX, β2−/− showed a 64% decrease in ϵPKC (p<0.01) and 22% increase in δPKC (p<0.01). The ϵPKC activator ΨϵRACK decreased mortality by 40% in β2−/− mice receiving DOX; there was no improvement in survival with the δPKC inhibitor δV1–1. After DOX, AKT activity was decreased by 76% (p<0.01) in β2−/− but not in WT. The α1-AR blocker prazosin, inhibiting signaling through Gαq, restored AKT activity and reduced DOX mortality by 47%. We next assessed the role of mitochondrial dysfunction in β2−/− mediated DOX toxicity. DOX treated β2−/− mice, but not WT, show marked vacuolization of mitochondrial cristae. Complex I activity decreased 31% in β2−/− mice with DOX; but not in WT. Baseline rate of Ca2+ release and peak [Ca2+]i ratio were increased 85% and 17% respectively in β2−/− myocytes compared to WT. Verapamil decreased mortality by 27% in DOX treated β2−/− mice. Cyclosporine, a blocker of both MPT and calcineurin, reduced DOX mortality to 50%. In contrast, FK506, a blocker of calcineurin but not the MPT, did not reduce DOX mortality. Cyclosporine prevented the decrease in AKT activity in β2−/− whereas FK506 did not. These findings suggest that β2-ARs modulate pro-survival kinases and attenuate mitochondrial dysfunction during DOX cardiotoxicity; absence of β2-ARs enhances DOX toxicity via negative regulation of survival kinases and enhancement of intracellular Ca2+, sensitizing mitochondria to opening of the MPT.


2015 ◽  
Vol 43 (4) ◽  
pp. 553-558 ◽  
Author(s):  
Ryan J. Middleton ◽  
Guo-Jun Liu ◽  
Richard B. Banati

The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands.


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