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KCNMA1 encoded cardiac BK channels afford protection against ischemia-reperfusion injury.

Soltysinska E, Bentzen BH, Barthmes M, Hattel H, Thrush AB, Harper ME, Qvortrup K, Larsen FJ, Schiffer TA, Losa-Reyna J, Straubinger J, Kniess A, Thomsen MB, Brüggemann A, Fenske S, Biel M, Ruth P, Wahl-Schott C, Boushel RC, Olesen SP, Lukowski R - PLoS ONE (2014)

Bottom Line: However, some studies challenged these cardio-protective roles of mitoBKs.In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation.While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP.

View Article: PubMed Central - PubMed

Affiliation: The Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark; Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

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Mitochondrial respiratory responses in mouse left heart ventricular fibres.O2-consumption of permeabilized BK+/+ fibres (blue bars) and fibres isolated from BK−/− hearts (red bars) were measured by applying an in vitro model of I/R at normoxia and reo-xygenation (both at 21% O2) upon 60 min of anoxia (by a N2 gassing system). For a representative original recording trace please refer to Figure S2 in File S1. (A) Coupled state 3 respiratory rate (OXPHOS) with complex I (CI) substrates malate (2 mmol/l) and glutamate (10 mmol/l) in the presence of ADP (5 mmol/l). (B) Cumulative OXPHOS of CI and II upon subsequent addition of succinate (10 mmol/l) to (A) for a supply of electrons to complex II (CII). OXPHOS of CI and II after 5 min (OXPHOS5) and after 60 min (OXPHOS60) of anoxia are shown. (C) Ratio of respiration upon addition of cytochrome c (CYTC) 5 min after 60 min anoxia compared to steady state respiratory flux rate before anoxia as a test for intactness of the IMM. (D) Isolated activity of complex IV (cytochrome c oxidase (COX)) with redox substrates ascorbate (2 mmol/l) and TMPD (5 mmol/l) in the presence of complex III blocker Antimycin A (2 µmol/l). (E) Ratio of OXPHOS to COX activity under fully oxygenated conditions showing loss of COX excess capacity in BK−/− as the ratio approaches 1.0 revealing an insufficient reserve capacity to produce energy in mitoBK-deficient fibres. Data are mean ± SEM with n = 6 for BK+/+ and n = 9 for BK−/− with ns = non-significant and **P<0.01 significantly different compared to BK+/+ (Fig. 2A, C, D two-tailed paired t-test). *P<0.05, **P<0.01, significantly different compared to BK+/+ within the respective condition (Fig. 2B).
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pone-0103402-g002: Mitochondrial respiratory responses in mouse left heart ventricular fibres.O2-consumption of permeabilized BK+/+ fibres (blue bars) and fibres isolated from BK−/− hearts (red bars) were measured by applying an in vitro model of I/R at normoxia and reo-xygenation (both at 21% O2) upon 60 min of anoxia (by a N2 gassing system). For a representative original recording trace please refer to Figure S2 in File S1. (A) Coupled state 3 respiratory rate (OXPHOS) with complex I (CI) substrates malate (2 mmol/l) and glutamate (10 mmol/l) in the presence of ADP (5 mmol/l). (B) Cumulative OXPHOS of CI and II upon subsequent addition of succinate (10 mmol/l) to (A) for a supply of electrons to complex II (CII). OXPHOS of CI and II after 5 min (OXPHOS5) and after 60 min (OXPHOS60) of anoxia are shown. (C) Ratio of respiration upon addition of cytochrome c (CYTC) 5 min after 60 min anoxia compared to steady state respiratory flux rate before anoxia as a test for intactness of the IMM. (D) Isolated activity of complex IV (cytochrome c oxidase (COX)) with redox substrates ascorbate (2 mmol/l) and TMPD (5 mmol/l) in the presence of complex III blocker Antimycin A (2 µmol/l). (E) Ratio of OXPHOS to COX activity under fully oxygenated conditions showing loss of COX excess capacity in BK−/− as the ratio approaches 1.0 revealing an insufficient reserve capacity to produce energy in mitoBK-deficient fibres. Data are mean ± SEM with n = 6 for BK+/+ and n = 9 for BK−/− with ns = non-significant and **P<0.01 significantly different compared to BK+/+ (Fig. 2A, C, D two-tailed paired t-test). *P<0.05, **P<0.01, significantly different compared to BK+/+ within the respective condition (Fig. 2B).

Mentions: Data are expressed as mean ± SEM. Electrophysiological results of BK+/+ were tested by a one-way ANOVA, and compared to BK−/− by a two-way ANOVA (Fig. 1). Electrophysiological experiments were treated as Bernoulli experiments. The effect of genotype on respiration and ROS production was evaluated using un-paired t-test (Fig. 2, Fig. 3 and Figure S3 in File S1) and a Two-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparison test (Fig. 2B). Infarct size was compared using One-way analysis of variance (ANOVA) test followed by a Turkey’s multiple comparison test (Fig. 4B). Morphometric characteristic and basal coronary flow and heart rate in 4 experimental groups was analyzed using One-way ANOVA test (Table S1 in File S1). Effects of the experimental protocols and genotypes on post-ischemic coronary flow and heart rate were compared using Two-way ANOVA followed by Bonferroni post-hoc test (Fig. 4C and D). Total reactive hyperemic volume was compared using an un-paired t-test (Figure S4 in File S1). Errors estimate was done by calculating the variability across different animals. Probability value of P<0.05 was considered significant.


KCNMA1 encoded cardiac BK channels afford protection against ischemia-reperfusion injury.

Soltysinska E, Bentzen BH, Barthmes M, Hattel H, Thrush AB, Harper ME, Qvortrup K, Larsen FJ, Schiffer TA, Losa-Reyna J, Straubinger J, Kniess A, Thomsen MB, Brüggemann A, Fenske S, Biel M, Ruth P, Wahl-Schott C, Boushel RC, Olesen SP, Lukowski R - PLoS ONE (2014)

Mitochondrial respiratory responses in mouse left heart ventricular fibres.O2-consumption of permeabilized BK+/+ fibres (blue bars) and fibres isolated from BK−/− hearts (red bars) were measured by applying an in vitro model of I/R at normoxia and reo-xygenation (both at 21% O2) upon 60 min of anoxia (by a N2 gassing system). For a representative original recording trace please refer to Figure S2 in File S1. (A) Coupled state 3 respiratory rate (OXPHOS) with complex I (CI) substrates malate (2 mmol/l) and glutamate (10 mmol/l) in the presence of ADP (5 mmol/l). (B) Cumulative OXPHOS of CI and II upon subsequent addition of succinate (10 mmol/l) to (A) for a supply of electrons to complex II (CII). OXPHOS of CI and II after 5 min (OXPHOS5) and after 60 min (OXPHOS60) of anoxia are shown. (C) Ratio of respiration upon addition of cytochrome c (CYTC) 5 min after 60 min anoxia compared to steady state respiratory flux rate before anoxia as a test for intactness of the IMM. (D) Isolated activity of complex IV (cytochrome c oxidase (COX)) with redox substrates ascorbate (2 mmol/l) and TMPD (5 mmol/l) in the presence of complex III blocker Antimycin A (2 µmol/l). (E) Ratio of OXPHOS to COX activity under fully oxygenated conditions showing loss of COX excess capacity in BK−/− as the ratio approaches 1.0 revealing an insufficient reserve capacity to produce energy in mitoBK-deficient fibres. Data are mean ± SEM with n = 6 for BK+/+ and n = 9 for BK−/− with ns = non-significant and **P<0.01 significantly different compared to BK+/+ (Fig. 2A, C, D two-tailed paired t-test). *P<0.05, **P<0.01, significantly different compared to BK+/+ within the respective condition (Fig. 2B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114839&req=5

pone-0103402-g002: Mitochondrial respiratory responses in mouse left heart ventricular fibres.O2-consumption of permeabilized BK+/+ fibres (blue bars) and fibres isolated from BK−/− hearts (red bars) were measured by applying an in vitro model of I/R at normoxia and reo-xygenation (both at 21% O2) upon 60 min of anoxia (by a N2 gassing system). For a representative original recording trace please refer to Figure S2 in File S1. (A) Coupled state 3 respiratory rate (OXPHOS) with complex I (CI) substrates malate (2 mmol/l) and glutamate (10 mmol/l) in the presence of ADP (5 mmol/l). (B) Cumulative OXPHOS of CI and II upon subsequent addition of succinate (10 mmol/l) to (A) for a supply of electrons to complex II (CII). OXPHOS of CI and II after 5 min (OXPHOS5) and after 60 min (OXPHOS60) of anoxia are shown. (C) Ratio of respiration upon addition of cytochrome c (CYTC) 5 min after 60 min anoxia compared to steady state respiratory flux rate before anoxia as a test for intactness of the IMM. (D) Isolated activity of complex IV (cytochrome c oxidase (COX)) with redox substrates ascorbate (2 mmol/l) and TMPD (5 mmol/l) in the presence of complex III blocker Antimycin A (2 µmol/l). (E) Ratio of OXPHOS to COX activity under fully oxygenated conditions showing loss of COX excess capacity in BK−/− as the ratio approaches 1.0 revealing an insufficient reserve capacity to produce energy in mitoBK-deficient fibres. Data are mean ± SEM with n = 6 for BK+/+ and n = 9 for BK−/− with ns = non-significant and **P<0.01 significantly different compared to BK+/+ (Fig. 2A, C, D two-tailed paired t-test). *P<0.05, **P<0.01, significantly different compared to BK+/+ within the respective condition (Fig. 2B).
Mentions: Data are expressed as mean ± SEM. Electrophysiological results of BK+/+ were tested by a one-way ANOVA, and compared to BK−/− by a two-way ANOVA (Fig. 1). Electrophysiological experiments were treated as Bernoulli experiments. The effect of genotype on respiration and ROS production was evaluated using un-paired t-test (Fig. 2, Fig. 3 and Figure S3 in File S1) and a Two-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparison test (Fig. 2B). Infarct size was compared using One-way analysis of variance (ANOVA) test followed by a Turkey’s multiple comparison test (Fig. 4B). Morphometric characteristic and basal coronary flow and heart rate in 4 experimental groups was analyzed using One-way ANOVA test (Table S1 in File S1). Effects of the experimental protocols and genotypes on post-ischemic coronary flow and heart rate were compared using Two-way ANOVA followed by Bonferroni post-hoc test (Fig. 4C and D). Total reactive hyperemic volume was compared using an un-paired t-test (Figure S4 in File S1). Errors estimate was done by calculating the variability across different animals. Probability value of P<0.05 was considered significant.

Bottom Line: However, some studies challenged these cardio-protective roles of mitoBKs.In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation.While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP.

View Article: PubMed Central - PubMed

Affiliation: The Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark; Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Show MeSH
Related in: MedlinePlus