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

Ewa Soltysinska; Bo Hjorth Bentzen; Maria Barthmes; Helle Hattel; A Brianne Thrush; Mary-Ellen Harper; Klaus Qvortrup; Filip J Larsen; Tomas A Schiffer; Jose Losa-Reyna; Julia Straubinger; Angelina Kniess; Morten Bækgaard Thomsen; Andrea Brüggemann; Stefanie Fenske; Martin Biel; Peter Ruth; Christian Wahl-Schott; Robert Christopher Boushel; Søren-Peter Olesen; Robert Lukowski

doi:10.1371/journal.pone.0103402

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

PLoS One. 2014 Jul 29;9(7):e103402. doi: 10.1371/journal.pone.0103402. eCollection 2014.

Authors

Ewa Soltysinska¹, Bo Hjorth Bentzen², Maria Barthmes³, Helle Hattel⁴, A Brianne Thrush⁵, Mary-Ellen Harper⁵, Klaus Qvortrup⁶, Filip J Larsen⁷, Tomas A Schiffer⁷, Jose Losa-Reyna⁷, Julia Straubinger⁸, Angelina Kniess⁸, Morten Bækgaard Thomsen¹, Andrea Brüggemann⁹, Stefanie Fenske¹⁰, Martin Biel¹⁰, Peter Ruth⁸, Christian Wahl-Schott¹⁰, Robert Christopher Boushel⁴, Søren-Peter Olesen¹, Robert Lukowski⁸

Affiliations

¹ 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.
² The Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark.
³ Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität, Munich, Germany; Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany; Nanion Technologies GmbH, Munich, Germany.
⁴ Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
⁵ Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.
⁶ Department of Biomedical Sciences, Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
⁷ Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
⁸ Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen, Germany.
⁹ Nanion Technologies GmbH, Munich, Germany.
¹⁰ Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität, Munich, Germany; Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Hypoxia
Disease Models, Animal
Energy Metabolism
Indoles / pharmacology
Ischemic Preconditioning
Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / genetics
Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / metabolism*
Large-Conductance Calcium-Activated Potassium Channels / chemistry
Large-Conductance Calcium-Activated Potassium Channels / genetics
Large-Conductance Calcium-Activated Potassium Channels / metabolism*
Membrane Potential, Mitochondrial / drug effects
Mice
Mice, Inbred C57BL
Mice, Knockout
Mitochondria, Heart / metabolism*
Muscle Fibers, Skeletal / ultrastructure
Muscle, Skeletal / metabolism
Myocardium / metabolism
Myocytes, Cardiac / cytology
Myocytes, Cardiac / drug effects
Myocytes, Cardiac / metabolism
Oxidative Phosphorylation / drug effects
Reactive Oxygen Species / metabolism
Reperfusion Injury / metabolism
Reperfusion Injury / pathology*
Tetrazoles / pharmacology
Thiourea / analogs & derivatives
Thiourea / pharmacology

Substances

1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-bromo-2-(1H-tetrazol-5-yl)phenyl)thiourea
Indoles
Kcnma1 protein, mouse
Large-Conductance Calcium-Activated Potassium Channel alpha Subunits
Large-Conductance Calcium-Activated Potassium Channels
Reactive Oxygen Species
Tetrazoles
paxilline
Thiourea

Grants and funding

This study was supported by the Danish National Research Foundation to ES, BHB and SPO, by the Danish Heart Foundation to BHB, by the Danish Agency for Science, Technology and Innovation, Medical Research Council (grant 10-084244) to MBT, and by Aase and Ejnar Danielsens Foundation and by Carlsberg Foundation to SPO and ES. CWS and MB received funding from the Bayerische Forschungsstiftung and from the SME innovation program “ZIM” of the German Federal Ministry of Economics and Technology (BMWi). RL and PR received grants from the Deutsche Forschungsgemeinschaft and acknowledge the support by the Open Access Publishing Fund of Tuebingen University. RL is a fellow of the Eliteprogramm of the Baden-Württemberg Stiftung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.