Flavonoids as new regulators of mitochondrial potassium channels: contribution to cardioprotection.

OBJECTIVES: Acute myocardial ischemia is one of the major causes of illness in western society. Reduced coronary blood supply leads to cell death and loss of cardiomyocyte population, resulting in serious and often irreversible consequences on myocardial function. Mitochondrial potassium (mitoK) channels have been identified as fine regulators of mitochondrial function and, consequently, in the metabolism of the whole cell, and in the mechanisms underlying the cardioprotection. Interestingly, mitoK channels represent a novel putative target for treating cardiovascular diseases, particularly myocardial infarction, and their modulators represent an interesting tool for pharmacological intervention. In this review, we took up the challenge of selecting flavonoids that show cardioprotective properties through the activation of mitoK channels. KEY FINDINGS: A brief overview of the main information on mitoK channels and their participation in the induction of cytoprotective processes was provided. Then, naringenin, quercetin, morin, theaflavin, baicalein, epigallocatechin gallate, genistein, puerarin, luteolin and proanthocyanidins demonstrated to be effective modulators of mitoK channels activity, mediating many beneficial effects. SUMMARY: The pathophysiological role of mitoK channels has been investigated as well as the impact of flavonoids on this target with particular attention to their potential role in the prevention of cardiovascular disorders.

Luteolin-Induced Activation of Mitochondrial BKCa Channels: Undisclosed Mechanism of Cytoprotection.

Luteolin (LUT) is a well-known flavonoid that exhibits a number of beneficial properties. Among these, it shows cardioprotective effects, as confirmed by numerous studies. However, its effect on mitochondrial potassium channels, the activation of which is related to cytoprotection, as well as on heart ischemia/reperfusion (I/R) damage prevention, has not yet been investigated. The large conductance calcium-regulated potassium channel (mitoBKCa) has been identified in both the mitochondria of the vascular endothelial cells, which plays a significant role in the functioning of the cardiovascular system under oxidative stress-related conditions, and in the mitochondria of cardiomyocytes, where it is deeply involved in cardiac protection against I/R injury. Therefore, the aim of this study was to explore the role of the mitoBKCa channel in luteolin-induced cytoprotection. A number of in vitro, in vivo, ex vivo and in silico studies have confirmed that luteolin activates this channel in the mitochondria of cardiomyocytes and endothelial cells, which in turn leads to the protection of the endothelium and a significant reduction in the extent of damage resulting from myocardial infarction, where this effect was partially abolished by the mitoBKCa channel blocker paxilline. In conclusion, these results suggest that luteolin has cardioprotective effects, at least in part, through the activation of the mitoBKCa channel, shedding light on a new putative mechanism of action.

Cytoprotective effects of the flavonoid quercetin by activating mitochondrial BKCa channels in endothelial cells.

Mitochondrial potassium channels have been implicated in cytoprotective mechanisms. Activation of the mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channel is important for protecting brain tissue against stroke damage as well as heart tissue against ischemia damage. In this paper, we examine the effect of the natural flavonoid quercetin as an activator of the mitoBKCa channel. Quercetin has a beneficial effect on many processes in the human body and interacts with many receptors and signaling pathways. We found that quercetin acts on mitochondria as a mitoBKCa channel opener. The activation observed with the patch-clamp technique was potent and increased the channel open probability from approximately 0.35 to 0.95 at + 40 mV in the micromolar concentration range. Moreover, quercetin at a concentration of 10 µM protected cells by reducing damage from treatment factors (tumor necrosis factor α and cycloheximide) by 40%, enhancing cellular migration and depolarizing the mitochondrial membrane. Moreover, the presence of quercetin increased the gene expression and protein level of the mitoBKCa ß3 regulatory subunit. The observed cytoprotective effects suggested the involvement of BKCa channel activation. Additionally, the newly discovered mitoBKCa activator quercetin elucidates a new mitochondrial pathway that is beneficial for vascular endothelial cells.

Identification of the Large-Conductance Ca2+-Regulated Potassium Channel in Mitochondria of Human Bronchial Epithelial Cells.

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca2+-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BKCa channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary ß3-ß4 subunits of BKCa channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately ß3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBKCa channel in a bronchial epithelial cell line were described.

Patch-Clamp Recording of the Activity of Ion Channels in the Inner Mitochondrial Membrane.

Mitochondria are intracellular organelles, which play a crucial role in the generation of ATP. Mitochondria are surrounded by a double membrane, consisting of a smooth outer membrane (OMM) and a markedly folded inner mitochondrial membrane (IMM). Mitochondrion that has been stripped of its outer membrane, leaving the inner membrane intact is called mitoplast. There is a number of different transport proteins located in the inner mitochondrial membrane including ion channels that mediate fluxes of potassium, calcium, and chloride ions. These channels regulate the mitochondrial membrane potential, respiration, and production of reactive oxygen species. The stability of mitoplasts offers the possibility of measuring the activity of ion channels from IMM using the patch-clamp technique. Electrophysiological measurements of currents through ion channels in the IMM permit discovery of unique properties of these channels with the aim of new specific pharmacological therapies. In this chapter, we describe the isolation of mitochondria, preparation of mitoplast for patch-clamp recordings and single-mitoplast PCR experiments, which can be helpful in mastering the technique of recording the activity of mitochondrial ion channels.

Regulation of the Mitochondrial BKCa Channel by the Citrus Flavonoid Naringenin as a Potential Means of Preventing Cell Damage.

Naringenin, a flavanone obtained from citrus fruits and present in many traditional Chinese herbal medicines, has been shown to have various beneficial effects on cells both in vitro and in vivo. Although the antioxidant activity of naringenin has long been believed to be crucial for its effects on cells, mitochondrial pathways (including mitochondrial ion channels) are emerging as potential targets for the specific pharmacological action of naringenin in cardioprotective strategies. In the present study, we describe interactions between the mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa channel) and naringenin. Using the patch-clamp method, we showed that 10 µM naringenin activated the mitoBKCa channel present in endothelial cells. In the presence of 30 µM Ca2+, the increase in the mitoBKCa channel probability of opening from approximately 0.25 to 0.50 at -40 mV was observed. In addition, regulation of the mitoBKCa channel by naringenin was dependent on the concentration of calcium ions. To confirm our data, physiological studies on the mitochondria were performed. An increase in oxygen consumption and a decrease in membrane potential was observed after naringenin treatment. In addition, contributions of the mitoBKCa channel to apoptosis and necrosis were investigated. Naringenin protected cells against damage induced by tumor necrosis factor (TNF-) in combination with cycloheximide. In this study, we demonstrated that the flavonoid naringenin can activate the mitoBKCa channel present in the inner mitochondrial membrane of endothelial cells. Our studies describing the regulation of the mitoBKCa channel by this natural, plant-derived substance may help to elucidate flavonoid-induced cytoprotective mechanisms.

Fenofibrate Reduces the Asthma-Related Fibroblast-To-Myofibroblast Transition by TGF-Β/Smad2/3 Signaling Attenuation and Connexin 43-Dependent Phenotype Destabilization.

The activation of human bronchial fibroblasts by transforming growth factor-ß1 (TGF-ß1) leads to the formation of highly contractile myofibroblasts in the process of the fibroblast⁻myofibroblast transition (FMT). This process is crucial for subepithelial fibrosis and bronchial wall remodeling in asthma. However, this process evades current therapeutic asthma treatment strategies. Since our previous studies showed the attenuation of the TGF-ß1-induced FMT in response to lipid-lowering agents (e.g., statins), we were interested to see whether a corresponding effect could be obtained upon administration of hypolipidemic agents. In this study, we investigated the effect of fenofibrate on FMT efficiency in populations of bronchial fibroblasts derived from asthmatic patients. Fenofibrate exerted a dose-dependent inhibitory effect on the FMT, even though it did not efficiently affect the expression of α-smooth muscle actin (α-SMA; marker of myofibroblasts); however, it considerably reduced its incorporation into stress fibers through connexin 43 regulation. This effect was accompanied by disturbances in the actin cytoskeleton architecture, impairments in the maturation of focal adhesions, and the fenofibrate-induced deactivation of TGF-ß1/Smad2/3 signaling. These data suggest that fenofibrate interferes with myofibroblastic differentiation during asthma-related subepithelial fibrosis. The data indicate the potential application of fenofibrate in the therapy and prevention of bronchial remodeling during the asthmatic process.

Mitochondrial potassium channels - an overview.

Mitochondria play a fundamental role in ATP synthesis within the majority of mammalian cells. Potassium channels present in the inner mitochondrial membrane are fine regulators of mitochondrial function, based on inner membrane K+ permeability. These channels are regulated by a plethora of factors and conditions in a way similar to plasma membrane potassium channels. Regulators of mitochondrial potassium channels include the membrane potential, calcium ions, free fatty acids and ATP levels within the cells. Recently, it was shown that these channels are regulated by the respiratory chain, stretching of the membrane and phosphorylation. The essential interest that has driven studies of mitochondrial potassium channels for nearly 25 years is their role in cytoprotection and in cell death. Mitochondrial potassium channels have been described in neurons, astrocytoma, cardiac and skeletal muscles, fibroblasts, keratinocytes and endothelial cells. In this overview, we summarize the current knowledge of mitochondrial potassium channels. This summary will be done with a special focus on studies performed over the last 20 years in the Laboratory of Intracellular Ion Channels at the Nencki Institute. These include studies on the electrophysiological and pharmacological properties of mitochondrial potassium channels and on their regulation by endogenous intracellular substances. Additionally, the regulation of mitochondrial potassium channels by the respiratory chain and by stretching of the inner mitochondrial membrane will be reviewed. Properties of mitochondrial potassium channels in various organisms will also be summarized.