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. 2016 Feb 5;11(2):e0148359.
doi: 10.1371/journal.pone.0148359. eCollection 2016.

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Lukas Jaroslaw Motloch  1 , Robert Larbig  1   2 , Tina Gebing  1 , Sara Reda  1 , Astrid Schwaiger  1 , Johannes Leitner  1 , Martin Wolny  1 , Lars Eckardt  2 , Uta C Hoppe  1
Affiliations

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Lukas Jaroslaw Motloch et al. PLoS One. .

Abstract

Introduction: The possible role of UCP2 in modulating mitochondrial Ca2+-uptake (mCa2+-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods: Thus, we analyzed mCa2+-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca2+-transients from mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2-/- mice.

Results: Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake, which was significantly decreased in UCP2-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2-/- cardiomyocytes (Ca2+)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca2+)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-.

Conclusion: Our results indicate regulatory effects of UCP2 on mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
a) Original traces of recordings of Ca2+-uptake in rhod2-AM loaded isolated cardiac mitochondria from WT and UCP2-/- after a bolus of 200 nM Ca2. b) Amplitude histogram of basic mCa1 in mitoplasts from WT. Basic mCa1 in WT showed three amplitude levels with -1.16±0.02 pA being the most common observed amplitude [Iunitary: -1.16±0.02 pA, n = 30; μ1: -1.16 pA (65%), μ2: -1.71 pA (31%), μ3: -3.10 pA (4%)]. c) In cardiac mitoplasts from WT Ru360 (200 nM) in PS (n = 8) and Ru360 (10μM) in BS (n = 5) as well as ATP (1mM) in BS (n = 5) decreased total open probability (Po,total) of mCa1 (*p<0.05). d) Examples of consecutive original traces of cardiac mCa1 in mitoplasts from WT: mCa1 in WT vs. mCa1 in WT + Ru360 (200 nM) in pipette solution (PS) vs. mCa1 in WT + Ru360 (10 μM) in bath solution (BS) vs. mCa1 in WT + ATP (1mM) in BS. e) Slope conductance of mCa1 in WT (12.59±1.57 pS, n = 17) and of mCa1 in UCP2-/- (12.79±1.83 pS, n = 13) was not different.
Fig 2
Fig 2
a) Examples of consecutive original traces of cardiac mCa1 in mitoplasts from UCP2-/-: mCa1 in UCP2-/- vs. mCa1 in UCP2-/- + Ru360 (200 nM) in PS vs. mCa1 in UCP2-/- + Ru360 (10 μM) in BS vs. mCa1 in UCP2-/- + ATP (1 mM) in BS. b) Amplitude histogram of basic mCa1 in cardiac UCP2-/- mitoplasts. Basic mCa1 in UCP2-/- showed three amplitude levels with -1.19±0.04 pA being the most common observed amplitude [Iunitary: -1.19±0.04 pA, n = 23; μ1: -1.16 pA (78%), μ2: -1.95 pA (21%), μ3: -3.51 pA (1%)]. c) In cardiac mitoplasts from UCP2-/- Ru360 (200 nM) in pipette solution (PS, n = 7) and Ru360 (10 μM) in bath solution (BS, n = 4) significantly decreased total open probability (Po, total) of mCa1 (*p<0.05). ATP (1 mM, n = 7) had no effect on mCa1 activity in UCP2-/-. d) Total open probabilities (Po, total) of basic mCa1: In comparison to WT mCa1, Po,total of mCa1 in UCP2-/- was significantly decreased (*p<0.05). e-g) Ca2+ transients in WT cardiomyocytes (Con) ± 10 and 100 nM Ru360. e) Representative traces of (Ca2+)c (left) and (Ca2+)m (right), f-g) Statistical analysis: In WT cardiomyocytes 10 nM Ru360 (10Ru) significantly elevated amplitude while (Ca2+)c rate of rise was decreased. 100 nM Ru360 (100Ru) suppressed both parameters. In mitochondria amplitude and rate of rise of (Ca2+)m were significantly decreased using either 10 or 100 nM Ru360. *p<0.05 vs. Con; #p<0.05 vs. Con + 10 nM Ru360.
Fig 3
Fig 3
a-c) Ca2+ transients in wild-type (WT) and in UCP2-/- cardiomyocytes (UCP2). a-b) Statistical analysis of (Ca2+)c (left) and (Ca2+)m (right) and c) representative traces of (Ca2+)c (left) and (Ca2+)m (right): In UCP2-/- cardiomyocytes rate of rise of (Ca2+)c was significantly decreased while the amplitude of (Ca2+)c was unchanged vs. WT (*p<0.05). The amplitude and rate of rise of (Ca2+)m of UCP2-/- cardiomyocytes were significantly decreased vs. WT (*p<0.05). d-f) Transsarcolemmal Ca2+ transients in WT vs. UCP2-/- cardiomyocytes evoked in the presence of 0.2 μM ryanodine, 0.01 mM thapsigargin and 100 nM Ru360. d) Representative traces of transsarcolemmal (Ca2+)c in WT and UCP2-/-, and e-f) Statistical analysis of amplitude (e) and rate of rise (f): In UCP2-/- transsarcolemmal Ca2+ transient amplitude and rate of rise were significantly down-regulated (*p<0.05 vs. WT). g) INCX WT vs. UCP2-/- cardiomyocytes: forward INCX was elicited by 1 s of exposure to 5 mM caffeine using a holding potential of -40 mV. No difference in caffeine induced INCX in WT compared to UCP2-/- was found. h) SR Ca2+ load in WT vs. UCP2-/- calculated via the integral of INCX recordings. No difference in SR Ca2+ load between WT and UCP2-/- was detected.
Fig 4
Fig 4
a-c) Ca2+ transients in UCP2-/- cardiomyocytes (UCP2) ± 10 and 100 nM Ru360. a) Representative traces of (Ca2+)c (left) and (Ca2+)m (right), and b-c) Statistical analysis: 10 nM Ru360 (10Ru) had no impact on either (Ca2+)c or (Ca2+)m transient amplitudes of UCP2-/- cardiomyocytes. 100 nM Ru360 (100Ru) significantly decreased the amplitude in both compartments without affecting the rate of rise. *p<0.05 vs. Con; #p<0.05 vs. Con + 10 nM Ru360. d-f) Ca2+ transients in WT cardiomyocytes + 5 (Con), + 0 (0ATP) and + 15 mM ATP (15ATP). d-e) Statistical analysis, and f) representative traces of (Ca2+)c (left) and (Ca2+)m (right): In the absence of ATP the amplitude of (Ca2+)m was significantly increased, while it was suppressed when ATP was elevated to 15 mM. Conversely, the amplitude of (Ca2+)c was significantly decreased in the absence of ATP and significantly increased with 15 mM ATP. The rate of rise of (Ca2+)m and (Ca2+)c was significantly decreased in the presence of 15 mM ATP. This inhibitory effect of ATP suggests a similar shift of dyadic cleft Ca2+ towards the ryanodine receptor (RyR) as in WT cardiomyocytes ± Ru360. The opposite effect was observed in the absence of ATP. *p<0.05 vs. Con; #p<0.05 vs. 15 mM ATP. g-i) Ca2+ transients in UCP2-/- cardiomyocytes + 5 (Con), + 0 (0ATP) and + 15 mM ATP (15ATP). g) Representative traces of (Ca2+)c (left) and (Ca2+)m (right), and h-i) statistical analysis: The modulation of the ATP concentration had no significant impact on either amplitude or rate of rise of Ca2+ in both cellular compartments.
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References

    1. Jouaville LS, Pinton P, Bastianutto C, Rutter GA, Rizzuto R. Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming. Proc Natl Acad Sci U S A. 1999;96(24):13807–12. Epub 1999/11/26. . - PMC - PubMed
    1. Territo PR, Mootha VK, French SA, Balaban RS. Ca(2+) activation of heart mitochondrial oxidative phosphorylation: role of the F(0)/F(1)-ATPase. Am J Physiol Cell Physiol. 2000;278(2):C423–35. Epub 2000/02/09. . - PubMed
    1. O'Rourke B. Mitochondrial ion channels. Annu Rev Physiol. 2007;69:19–49. Epub 2006/10/25. 10.1146/annurev.physiol.69.031905.163804 . - DOI - PMC - PubMed
    1. Kirichok Y, Krapivinsky G, Clapham DE. The mitochondrial calcium uniporter is a highly selective ion channel. Nature. 2004;427(6972):360–4. Epub 2004/01/23. 10.1038/nature02246 [pii]. . - DOI - PubMed
    1. Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, et al. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science. 1998;280(5370):1763–6. Epub 1998/06/20. . - PubMed

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This work was supported by grants from the Marga and Walter Boll-Stiftung (UCH) and the Paracelsus Medical University, Salzburg (R-12/04038-LAR; LJM and RL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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