Blue Native Electrophoresis Research Articles

How hot can mitochondria be? Incubation at temperatures above 43 °C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria

Mitochondrial function generates an important fraction of the heat that contributes to cellular and organismal temperature maintenance, but the actual values of this parameter reached in the organelles is a matter of debate. The studies addressing this issue have reported divergent results: from detecting in the organelles the same temperature as the cell average or the incubation temperature, to increasing differences of up to 10 degrees above the incubation value. Theoretical calculations based on physical laws exclude the possibility of relevant temperature gradients between mitochondria and their surroundings. These facts have given rise to a conundrum or paradox about hot mitochondria.We have examined by Blue-Native electrophoresis, both in intact cells and in isolated organelles, the stability of respiratory complexes and supercomplexes at different temperatures to obtain information about their tolerance to heat stress. We observe that, upon incubation at values above 43 °C and after relatively short periods, respiratory complexes, and especially complex I and its supercomplexes, are unstable even when the respiratory activity is inhibited. These results support the conclusion that high temperatures (>43 °C) cause damage to mitochondrial structure and function and question the proposal that these organelles can physiologically work at close to 50 °C.

Method for detecting CoQ10 incorporation in the mitochondrial respiratory chain supercomplex.

Coenzyme Q10 is an important component of the mitochondrial electron transfer chain. A supercomplex of mitochondrial electron transfer system proteins exists. This complex also contains coenzyme Q10. The concentrations of coenzyme Q10 in tissues decrease with age and pathology. Coenzyme Q10 is given as a supplement. It is unknown whether coenzyme Q10 is transported to the supercomplex. We develop a method for measuring coenzyme Q10 in the mitochondrial respiratory chain supercomplex in this study. Blue native electrophoresis was used to separate mitochondrial membranes. Electrophoresis gels were cut into 3 mm slices. Hexane was used to extract coenzyme Q10 from this slice, and HPLC-ECD was used to analyze coenzyme Q10. Coenzyme Q10 was found in the gel at the same site as the supercomplex. Coenzyme Q10 at this location was thought to be coenzyme Q10 in the supercomplex. We discovered that 4-nitrobenzoate, a coenzyme Q10 biosynthesis inhibitor, reduced the amount of coenzyme Q10 both within and outside the supercomplex. We also observed that the addition of coenzyme Q10 to cells increased the amount of coenzyme Q10 in the supercomplex. It is expected to analysis coenzyme Q10 level in supercomplex in various samples by using this novel method.

Assessing Oligomerization Status of Mitochondrial OXPHOS Complexes Via Blue Native Page.

Mitochondrial metabolism plays key roles in pathologies such as cancer. The five complexes of the oxidative phosphorylation (OXPHOS) system are crucial for producing ATP and maintaining cellular functions and are particularly exploited in cancer cells. Understanding the oligomeric state of these OXPHOS complexes will help elucidate their function (or dysfunction) in cancer cells and can be used as a mechanistic tool for anticancer agents that target mitochondria. Here we describe a protocol to observe the oligomeric state of the five OXPHOS complexes by isolating mitochondrial-enriched fractions followed by assessing their oligomeric state by nondenaturing blue native page electrophoresis.

High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes.

Blue native electrophoresis (BN-PAGE) is a highly resolutive method suited to the study of high molecular weight protein complexes between 100 and >3000kDa. One of the drawbacks of this method is that it is very time-consuming and requires high quantities of purified organelles. Here we describe a high throughput BN-PAGE method allowing to screen libraries of plants potentially altered in respiratory metabolism.

ATPAF1 deficiency impairs ATP synthase assembly and mitochondrial respiration

ATP11p and ATP12p are two nuclear-encoded mitochondrial chaperone proteins required for assembling the F1Fo-ATP synthase F1 sector. ATPAF1 and ATPAF2 are the mammalian homologs of ATP11p and ATP12p. However, the biochemical and physiological relevance of ATPAF1 and ATPAF2 in animal tissues with high energy-dependence remains unclear. To explore the in vivo role of ATP assembly and the effects of ATP synthase deficiency in animals, we have generated knockout (KO) mouse models of these assembly factors using CRISPR/Cas9 technology. While the Atpaf2-KO mice were embryonically lethal, Atpaf1-KO mice grew to adulthood but with smaller body sizes and elevated blood lactate later in life. We specifically investigated how ATPAF1 deficiency may affect ATP synthase biogenesis and mitochondrial respiration in the mouse heart, an organ highly energy-dependent. Western blots and Blue-Native electrophoresis (BN-PAGE) demonstrated a decreased F1 content and ATP synthase dimers in the Atpaf1-KO heart. Mitochondria from ATPAF1-deficient hearts showed ultrastructural abnormalities with condensed degenerated mitochondria, loss of cristae, and impaired respiratory capacity. ATP synthase deficiency also leads to impaired autophagy and mitochondrial dynamic. Consequently, decreased cardiac function was exhibited in adult Atpaf1-KO mice. The results provide strong support that ATPAF1 is essential for ATP synthase assembly and mitochondrial oxidative phosphorylation, thus playing a crucial role in maintaining cardiac structure and function in animals.

The role of <i>Arabidopsis thaliana</i> photoreceptors in regulating the process of state transitions

The initial formation of the photosynthetic apparatus in plants occurs during photomorphogenesis. The red/far-red (phytochromes) and blue (cryptochrome) light protein-photoreceptors play the most important role in photomorphogenesis initiation and regulation. The exited phytochrome and cryptochrome molecules can interact with transcription factors, changing the expression of nuclear genes, which encode the proteins of the plant photosynthetic apparatus. Since light is a variable factor, plants have developed appropriate adaptation mechanisms, including their photosynthetic apparatus protection. The mechanism of state transitions ensures a rapid adaptation of the photosynthetic apparatus. This adaptation mechanism increases the adsorption efficiency under current light conditions and prevents intensive generation of active forms of oxygen in chloroplasts, which leads to photo-oxidation and even cell death. This work aims to determine the role of photoreceptors - phytochromes A and B, as well as cryptochrome 1 and 2 - in regulating the process of state transitions in the Arabidopsis thaliana model plant. Arabidopsis mutants with the defects on A and B phytochromes and cryptochrome 1 and 2 genes were used as the research objects. The blue native electrophoresis in polyacrylamide gel was used to visualise state transitions. It was found that these photoreceptors had no direct effect on the redox-regulation of the state transitions mechanism in Arabidopsis. Presumably, these photoreceptors protect the photosynthetic apparatus from excessive light not by regulating the state transitions but indirectly, through regulating the chlorophyll, carotenoid and antioxidant components content.

The changes of PSII supercomplex stoichiometry in egy1 mutants are related to chlorophyll b deficiency

EGY1 is a chloroplast metalloprotease, the physiological role of which remains elusive. The changes observed in physiology and gene expression in egy1 mutants indicate that lack of the protease leads to yellow-green phenotype, changes in stoichiometry in PSII complexes, and early senescence. However, the knowledge concerning the role of the EGY1 to maintain the PSII function remains elusive. The aim of our study was to gain a deeper insight into the role of EGY1 protease in maintaining proper stoichiometry of PSII complexes. We applied the blue native electrophoresis technique as well as the immunoblotting method to investigate the abundance of PSII supercomplexes and selected individual PSII apoproteins in two Arabidopsis thaliana egy1 mutant lines. We also performed analyses of photosynthetic pigment content using DMSO assay. All analyses were performed in three biological replicates. Our results revealed reductions in contents of LHCII trimers and monomers in both egy1 mutant lines, as well as lower accumulation levels of Lhcb1 and Lhcb2 (but not Lhcb3) apoproteins. These changes were accompanied by an increased chlorophyll a/b ratio. We conclude that the observed pattern of changes in PSII stoichiometry is related to chlorophyll b deficiency. This reduction of chlorophyll b content is not, however, related to chlorophyllide a oxygenase abundance.

Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections.

Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.

SEC24 isoform specificity regulates the assembly of γ‐secretase from dimeric subcomplexes

AbstractBackgroundγ‐Secretase is a diaspartyl protease with presenilin (PSEN) as the catalytic subunit. It targets a plethora of type I transmembrane proteins and, as such, affects a broad range of physiological processes. Because of this, aberrant functioning of γ‐secretase is linked to several diseases including cancer and Alzheimer’s disease (AD). Herein, mutations in the PSEN genes are associated with rare cases of familial AD and shift the production to longer, more aggregation prone amyloid β peptides. γ‐Secretase consists, besides the catalytic component PSEN, of nicastrin, APH1 and PEN‐2 that are co‐translationally translocated in the endoplasmic reticulum (ER). As γ‐secretase has a stoichiometry of 1:1:1:1, the assembly process is spatially and temporally regulated. Although it is generally agreed that this occurs in early biosynthetic compartments, it remains far from understood whether full assembly occurs already in the ER and to what extent Golgi‐to‐ER recycling is involved.MethodWe used advanced cell fractionation protocols and an in vitro reconstituted ER budding assay to explore in detail the stepwise assembly process of γ‐secretase in early biosynthetic compartments.ResultCell fractionation combined with blue native electrophoresis shows that subcomplexes are mainly present in ER‐enriched fractions while full complexes appear in the ER‐Golgi intermediate compartment, indicating that final assembly occurs beyond the ER. In vitro reconstitution of ER export reveals that none of the γ‐secretase subunits is required for ER‐exit of the others. However, knock‐out of any subunit induces the accumulation of preceding subcomplexes in de novo formed COPII vesicles. Mutating a motif in the cytosolic loop domain of PSEN1, which is lost in the familial AD‐associated PSEN1∆E9 mutant, abrogates ER‐exit of PSEN1 as well as PEN‐2, but not of nicastrin. This is explained molecularly by the selectivity of Sec24A/B and Sec24C/D in packaging PSEN1 and nicastrin, respectively, and further argues against full complex assembly prior to ER‐exit.ConclusionIn conclusion, our data support a model wherein individual subunits and dimeric subcomplexes preferentially exit the ER. Full assembly likely occurs in intermediate compartment/cis‐Golgi allowing complexes to escape active Golgi‐to‐ER recycling as part of a secondary quality control mechanism in controlling cellular γ‐secretase levels and activity.

Production of dye-binding esterase reactor after separation and detection using combined native isoelectric focusing and blue native electrophoresis

Production of dye-binding esterase reactor after separation and detection using combined native isoelectric focusing and blue native electrophoresis

Analyzing electron transport chain supercomplexes.

This review focuses on three independent and complementary approaches to obtain information on the combined function of respiratory complexes when present in different structural situations, either as individual complexes or when superassembled with other complexes. We review the utility of in-gel activity after blue native electrophoresis, integrated oxygen consumption of supercomplexes containing complex IV, and spectrophotometric activity measurements.

Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase dimers and to the generation of the full-conductance mitochondrial megachannel

The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.

Mitochondrial cristae narrowing upon higher 2-oxoglutarate load

Hypoxia causes mitochondrial cristae widening, enabled by the ~20% degradation of Mic60/mitofilin, with concomitant clustering of the MICOS complex, reflecting the widening of crista junctions (outlets) (Plecitá-Hlavatá et al. FASEB J., 2016 30:1941–1957). Attempting to accelerate metabolism by the addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to HepG2 cells pre-adapted to hypoxia, we found cristae narrowing by transmission electron microscopy. Glycolytic HepG2 cells, which downregulate hypoxic respiration, instantly increased respiration with dm2OG. Changes in intracristal space (ICS) morphology were also revealed by 3D super-resolution microscopy using Eos-conjugated ICS-located lactamase-β. Cristae topology was resolved in detail by focused-ion beam/scanning electron microscopy (FIB/SEM). The spatial relocations of key cristae-shaping proteins were indicated by immunocytochemical stochastic 3D super-resolution microscopy (dSTORM), while analyzing inter-antibody-distance histograms: i) ATP-synthase dimers exhibited a higher fraction of shorter inter-distances between bound F1-α primary Alexa-Fluor-647-conjugated antibodies, indicating cristae narrowing. ii) Mic60/mitofilin clusters (established upon hypoxia) decayed, restoring isotropic random Mic60/mitofilin distribution (a signature of normoxia). iii) outer membrane SAMM50 formed more focused clusters. Less abundant fractions of higher ATP-synthase oligomers of hypoxic samples on blue-native electrophoresis became more abundant fractions at the high dm2OG load and at normoxia. This indicates more labile ATP-synthase dimeric rows established at crista rims upon hypoxia, strengthened at normoxia or dm2OG-substrate load. Hypothetically, the increased Krebs substrate load stimulates the cross-linking/strengthening of rows of ATP-synthase dimers at the crista rims, making them sharper. Crista narrowing ensures a more efficient coupling of proton pumping to ATP synthesis. We demonstrated that cristae morphology changes even within minutes.

Abstracts of poster presenters in alphabetical sequence of first author

Iron is a crucial nutrient for proliferating cancer cells. Thus, we have synthesized a mitochondrially targeted derivative of the iron chelator deferoxamine (mDFO) and evaluated its ability to selectively eliminate cancer cells both in vitro and in vivo. Viability of human cancer cells treated with mDFO was assessed by crystal violet or annexin V staining. ROS production was assessed through fluorescent probes MitoSOX and DCF. Blue native electrophoresis and Seahorse were used to analyse mitochondrial function. The in vivo effect of mDFO was assessed by xenografting triple negative breast cancer cells in NOD/Scid mice. Our results show that mDFO is at least 100-fold more efficient than DFO in killing cancer cells. In addition, mDFO was shown to reduce respiration and dramatically decrease the amount and composition of mitochondrial respiratory supercomplexes. Both the latter effects might trigger the observed increase in mitochondrial superoxide production, the fragmentation of mitochondria and the activation of mitophagy. Importantly, in vivo results show that mDFO is effective in slowing the tumour growth. mDFO represents an effective drug to inhibit tumour growth in vivo and might represent the lead compound in a new generation of anti-cancer agents based on mitochondrial iron chelation.

The Oligomeric State of the Plasma Membrane H⁺-ATPase from Kluyveromyces lactis.

The plasma membrane H+-ATPase was purified from the yeast K. lactis. The oligomeric state of the H+-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H+-ATPase hexamer in both MASs as the sole/main oligomeric state—in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H+-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H+-ATPase. It is concluded that the quaternary structure of the H+-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.

Proteomic Analysis of Arabidopsis pldα1 Mutants Revealed an Important Role of Phospholipase D Alpha 1 in Chloroplast Biogenesis.

Phospholipase D alpha 1 (PLDα1) is a phospholipid hydrolyzing enzyme playing multiple regulatory roles in stress responses of plants. Its signaling activity is mediated by phosphatidic acid (PA) production, capacity to bind, and modulate G-protein complexes or by interaction with other proteins. This work presents a quantitative proteomic analysis of two T-DNA insertion pldα1 mutants of Arabidopsis thaliana. Remarkably, PLDα1 knockouts caused differential regulation of many proteins forming protein complexes, while PLDα1 might be required for their stability. Almost one third of differentially abundant proteins (DAPs) in pldα1 mutants are implicated in metabolism and RNA binding. Latter functional class comprises proteins involved in translation, RNA editing, processing, stability, and decay. Many of these proteins, including those regulating chloroplast protein import and protein folding, share common functions in chloroplast biogenesis and leaf variegation. Consistently, pldα1 mutants showed altered level of TIC40 (a major regulator of protein import into chloroplast), differential accumulation of photosynthetic protein complexes and changed chloroplast sizes as revealed by immunoblotting, blue-native electrophoresis, and microscopic analyses, respectively. Our proteomic analysis also revealed that genetic depletion of PLDα1 also affected proteins involved in cell wall architecture, redox homeostasis, and abscisic acid signaling. Taking together, PLDα1 appears as a protein integrating cytosolic and plastidic protein translations, plastid protein degradation, and protein import into chloroplast in order to regulate chloroplast biogenesis in Arabidopsis.

Landscape of the <i>Plasmodium</i> Interactome

Malaria represents a major global health issue, and the identification and prioritisation of new intervention targets remains an urgent priority. This search is significantly hampered by the fact that over a third of the genes of the malaria-causing Plasmodium parasites remain uncharacterized. We report the first large-scale interaction map of de novo protein assemblies in Plasmodium parasites, generated by combining blue native polyacrylamide electrophoresis with quantitative mass spectrometry and machine learning. Our integrative approach, which spans three different Plasmodium species, identified over twenty thousand putative protein interactions in Plasmodium schizonts, the majority of which are novel, and organized them into 600 complexes. We validate selected novel interactions, thus assigning putative functions in chromatin regulation to previously unannotated proteins and suggesting a role for an EELM2 domain-containing protein and a putative microrchidia protein as mechanistic links between complexes of AP2-domain transcription factors and epigenetic regulation. This represents the first high-confidence map of the native organisation of core conserved cellular processes in Plasmodium parasites. The network reveals putative functions of currently uncharacterized malaria proteins, provides mechanistic and structural insight, and reveals potential new therapeutic targets by grouping them into complexes with known targets.

Identification of the Molecular Partners of Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) That Are Involved in Human Lymphocyte Activation

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein that is found in lymphocytes and is tightly associated with the phosphatase CD45. The function of LPAP is still unknown. Studies of the LPAP interactome may reveal new details of how C45 and lymphocyte signaling in general are regulated. LPAP binding partners were sought using coimmunoprecipitation coupled with mass spectrometry, stabilization of protein complexes with chemical crosslinkers, and Blue Native electrophoresis. In addition to CD45, several proteins were identified as LPAP partners, including CD71, CD98, cytoskeletal proteins, the amino acid transporter SLC1A4, and the cell signaling component HS1. It was confirmed that more than 70% of LPAP molecules were bound with CD45 in a 1 : 1 complex. The effect of CD45 on LPAP was studied in CEM and Jurkat cells with a CD45 knockout. The LPAP levels in the cells were 10% of the level in wild-type cells. In the absence of CD45, LPAP phosphorylation at Ser-153 and Ser-163 was not affected, whereas phosphorylation at Ser-99 and Ser-172 decreased significantly. Based on the results, CD45 was assumed to play a role in regulating LPAP expression and phosphorylation status.

Comparative 1D Blue-Native electrophoresis analysis of Plasmodium falciparum and human proteins associated with cytoadherence

BackgroundTo understand more about changes to the molecular components that occur when host endothelium interacts with Plasmodium falciparum-infected erythrocytes, a combined technique of protein separation (1D Blue-Native electrophoresis) and mass spectrometry of infected erythrocytes with endothelial cells (EC) in a co-culture system has been used.MethodsNative proteins were extracted from co-cultures and identified by mass spectrometry. Proteomic data from different parasite strains, either adhesion proficient (to endothelial cells) or non-adherent, were analysed in parallel to reveal protein associations linked to cytoadherence. Informatic approaches were developed to facilitate this comparison.ResultsBlue-Native gel separation and LC/MS/MS identification revealed major differences in samples produced from endothelial cell co-culture with adherent and non-adherent parasite strains. This approach enabled us to identify protein associations seen only with the adhesion proficient parasite strain.ConclusionsThe combination of proteomic and analytical approaches has identified differences between adherent and non-adherent parasite lines in co-culture with EC, providing potential candidates for complexes or associations formed during cytoadherence involved in cell structure, signalling and apoptosis.

Abstract 431: Upregulation of Small Conductance Ca 2+ Activated K + Channels Promotes Formation of Mitochondrial Supercomplexes and Increases Respiration Reserve

Introduction: Small conductance calcium (Ca 2+ )-activated K + (SK) channels are present in the inner mitochondria membrane of ventricular cardiomyocytes. We recently reported that pharmacological enhancement of mito-SKs protects from Ca 2+ dependent arrhythmia by reducing production of reactive oxygen species (ROS) by mitochondria thereby normalizing oxidation state and activity of Ca 2+ release channels ryanodine receptors in hypertrophic rat hearts. Recent advances demonstrate that respiratory components of the mitochondrial electron transport chain can organize into supercomplexes (SCs) resulting in more efficient electron transfer and reduced generation of ROS. Hypothesis: Functional upregulation of mito-SK channels protects from Ca 2+ -dependent arrhythmia via promotion of mitochondrial SC formation and improved respiration. Methods: H9C2 myoblasts were infected with adenoviral vectors to overexpress SK2 and Cox7rp. After 48 hours in culture, cell respiration was measured using Seahorse XFe Analyzer; ROS was measured using general ROS indicator CFDA; and mitochondria were isolated to assess SC formation using Blue-Native electrophoresis (BN-PAGE). The effects of SK enhancer NS309 (2 μM, 10 min.) on SC formation were studied in Langendorff perfused rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Results: rSK2 overexpression in H9C2s reduced intracellular ROS ~ 30%, enhanced maximum respiration rate vs. controls (0.74±0.04 and 0.58±0.03 pM/min/μg respectively, p<0.05); and caused a robust shift (>2 fold) of complex IV towards large mitochondrial SCs (~1.2 mDa). As a control, similar results were obtained with overexpression of Cox7rp protein that promotes SC formation. In addition, BN-PAGE experiments in isolated rat hearts demonstrated that robust decrease in high MW Complexes I and IV in hypertrophy was fully restored to control levels upon treatment with SK enhancer NS309. Conclusion: Upregulation of SK channels reduces mito-ROS by an increase in respiration reserve which is linked to enhanced mitochondrial SC formation. Our data suggest that targeting mito-SK channels has therapeutic potential to treat cardiac arrhythmia in conditions accompanied by oxidative stress.