BK channels as targets of cGMP signalling in myocardial pre- and postconditioning and survival

Robert Lukowski and Peter Ruth, Tübingen

The role of the NO/cGMP/cGMP-dependent protein kinase I (cGKI) pathway in myocardial protection and pre-/postconditioning has been intensively investigated. Drugs that block the activity of cGMP-degrading enzymes, such as the phosphodiesterase 5 inhibitor sildenafil, have a pre-conditioning-like effect on the survival of cardiomyocytes after ischaemia/reperfusion (I/R) injury and reduce infarction area. In cardiac myocytes, accumulation of cGMP leads to several protective events including stimulation of mitochondrial ATP-dependent potassium channels (mitoKATP) at the inner mitochondrial membrane (IMM). Increased K+ fluxes into the mitochondrial matrix causes a rise in reactive oxygen species production via the electron transport chain and an activation of the epsilon isoform of protein kinase C. Ca2+-activated K+ channels of the BK type, in cardiomyocytes exclusively present at the IMM, play also a role in protection against I/R injury. MitoBK channels significantly contribute to mitochondrial K+ uptake, and mitoBK openers have been shown to protect hearts against infarction. Importantly, knockdown of the accessory b1-subunit of the BK channel abolished the infarct limiting effects of the cGMP-elevating drug sildenafil. These findings indicate that mitoBK activation confers cardioprotection in a manner similar to but independent of mitoKATP activation and, together with the well-established signalling of NO/cGMP/cGKI to plasma membrane BK channels in smooth muscle cells and neurons, suggest a direct link between cGMP- and mitoBK-mediated effects in ischaemic conditioning. To investigate the role of mitoBK as a target of cGMP signalling in cardiac cells in vivo, we aim to study BK-/- mice with a global deletion in addition to mice with cardiomyocyte-specific inactivation of BK. The protection against I/R injury will be tested by ischaemic pre- and postconditioning, ligands of particulate guanylyl cyclase receptors, NO-stimulated guanylyl cyclases, and compounds that directly modulate BK activity. We will also assess the long-term outcome after occlusion and reperfusion of the left coronary artery by comparing infarct size and survival of BK knockout and litter-matched control mice. Analyses of cGMP pools at I/R and effects of cGMP on functional characteristics of mitochondria isolated from cardiomyocytes of gene-targeted BK mouse mutants together with approaches to reveal electro-pharmacological properties of mitoBK will corroborate the in vivo studies, and will finally improve our understanding of cGMP signalling to K+ channels at the IMM.

Fig. 1. Putative cGMP/K+ channel signalling in mitochondria of cardiomyocytes at ischaemia/reperfusion. Ischaemia and reperfusion injury lead to a series of detrimental events causing mitochondrial Ca2+ overload, particularly during reperfusion when oxygen is reintroduced and the IMM potential (ΔΨm) is regenerated. With the return of oxygen, a large burst of ROS has been consistently shown to occur, which leads to extensive oxidative damage of mitochondria. Both Ca2+ and ROS accumulation trigger the formation of the mitochondrial permeability transition pore (mPTP). mPTP opening leads to irreversible damages such as the collapse of the ΔΨm and ATP depletion, a cytotoxic burst of ROS to the cell and cardiomyocyte Ca2+ overload. Additionally, the changes in mitochondrial integrity cause a release of cytochrome C, which in turn induces a series of events leading to cell death. Protection by cGMP upon prolonged ischaemia of cardiomyocytes involves the activation of different targets. cGKI is probably the most important effector and may mediate transfer of signals, probably via yet unidentified protective molecules as indicated by X/Y at the outer membrane of the mitochondrium with access to the inter-membrane space (IMS). This cytosol-to-mitochondria signalling causes opening of mitoKATP (mKATP) (Costa et al. 2005) that modulate mitochondrial bioenergetics i.e. a depolarization of the IMM potential. In cardiomyocytes, BK channels have been identified exclusively at IMM (mBK) (Xu et al. 2002). Direct stimulation of canonical BK channel by cGMP/cGKI is well established (Swayze et al. 2001) in various cell types. In addition, BK openers such as NS1619 and NS11021, when applied at early onset of reperfusion, have been shown to protect the heart from injury (Bentzen et al. 2009; Wang et al. 2004). Matrix K+ influx via mitochondrial K+ channels inhibits Ca2+ overload during I/R and causes a mild uncoupling of the electron transport chain (ETC), which in turn triggers ROS formation at ischaemia. Limited ROS production prevents mPTP opening at reperfusion by a mechanism which might involve “survival” kinases such as PKC. However, it has never been tested in genetically modified mouse models whether cardioprotection afforded by cGMP depends on BK channels. Importantly, it is not clear whether cGMP pools generated by particulate guanylyl cyclase receptors (GC-A/B) and NO-stimulated guanylyl cyclases (NO-GC) signal equally well to the IMM at I/R. In addition, in vitro and in vivo data indicate that a NO/cGMP-dependent pathway controls mitochondrial biogenesis and stimulates mitochondrial function in different organs including the heart (Nisoli et al. 2004). (C) Robert Lukowski

Project-related publications

PIs of this project; PIs of other FOR 2060 projects are in bold.

1.    Straubinger J, Schöttle V, Bork N, Subramanian H, Dünnes H, Russwurm M, Gawaz M, Friebe A, Nemer M, Nikolaev VO, Lukowski R. Sildenafil does not prevent heart hypertrophy and fibrosis induced by cardiomyocyte AT1R signalling. J Pharmacol Exp Ther. 2015;354:406-16. [Project 3, Project 4, and Project 5] [pubmed]

2.    Brennenstuhl C, Tanimoto N, Burkard M, Wagner R, Bolz S, Trifunovic D, Kabagema-Bilan C, Paquet-Durand F, Beck SC, Huber G, Seeliger MW, Ruth P, Wissinger B, Lukowski R. Targeted ablation of the Pde6h gene in mice reveals cross-species differences in cone and rod phototransduction protein isoform inventory. J Biol Chem. 2015;290:10242-55. [Project 5 and Project 9] [pubmed]

3.    Lu R, Bausch AE, Kallenborn-Gerhardt W, Stoetzer C, Debruin N, Ruth P, Geisslinger G, Leffler A, Lukowski R*, Schmidtko A*. Slack channels expressed in sensory neurons control neuropathic pain in mice. J Neurosci. 2015;35:1125-35. (*equal contribution) [Project 5] [pubmed]

4.    Feil S, Fehrenbacher B, Lukowski R, Essmann F, Schulze-Osthoff K, Schaller M, Feil R. Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circ Res. 2014;115:662-7. [Project 1 and Project 5] [pubmed]

5.    Lu R, Lukowski R, Sausbier M, Zhang DD, Sisignano M, Schuh CD, Kuner R, Ruth P, Geisslinger G, Schmidtko A. BKCa channels expressed in sensory neurons modulate inflammatory pain in mice. Pain. 2014;155:556-65. [Project 5] [pubmed]

6.    Leiss V, Illison J, Domes K, Hofmann F, Lukowski R. Expression of cGMP-dependent protein kinase type I in mature white adipocytes. Biochem Biophys Res Commun. 2014;452:151-6. [Project 5] [pubmed]

7.    Patrucco E, Domes K, Sbroggio M, Blaich A, Schlossmann J, Desch M, Rybalkin SD, Beavo JA, Lukowski R, Hofmann F. Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis. Proc Natl Acad Sci U S A. 2014;111:12925-9. [Project 5] [pubmed]

8.    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, Bruggemann A, Fenske S, Biel M, Ruth P, Wahl-Schott C, Boushel RC, Olesen SP, Lukowski R. KCNMA1 encoded cardiac BK channels afford protection against ischaemia-reperfusion injury. PLoS One. 2014;9:e103402. [Project 5] [pubmed]

9.    Zhang L*, Lukowski R*, Gaertner F, Lorenz M, Legate KR, Domes K, Angermeier E, Hofmann F, Massberg S. Thrombocytosis as a response to high interleukin-6 levels in cGMP-dependent protein kinase I mutant mice. Arterioscler Thromb Vasc Biol. 2013;33:1820-8. (*equal contribution) [Project 5] [pubmed]

10.  Methner C, Lukowski R, Grube K, Loga F, Smith RA, Murphy MP, Hofmann F, Krieg T. Protection through postconditioning or a mitochondria-targeted S-nitrosothiol is unaffected by cardiomyocyte-selective ablation of protein kinase G. Basic Res Cardiol. 2013;108:337. [Project 5] [pubmed]