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The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca2+ handling, and actin--myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]i. Notably, diastolic [ATP]i was <1 mM, which is eightfold to 10-fold lower than previously estimated. Accordingly, ATP -sensitive K+ (KATP) channels were active at physiological [ATP]i. Cells exhibited two distinct types of ATP fluctuations during an action potential: net increases (Mode 1) or decreases (Mode 2) in [ATP]i. Mode 1 [ATP]i increases necessitated Ca2+ entry and release from the sarcoplasmic reticulum (SR) and were associated with increases in mitochondrial Ca2+. By contrast, decreases in mitochondrial Ca2+ accompanied Mode 2 [ATP]i decreases. Down-regulation of the protein mitofusin 2 reduced the magnitude of [ATP]i fluctuations, indicating that SR-mitochondrial coupling plays a crucial role in the dynamic control of ATP levels. Activation of ß-adrenergic receptors decreased [ATP]¡, underscoring the energetic impact of this signaling pathway. Finally, our work suggests that cross-bridge cycling is the largest consumer of ATP in a ventricular myocyte during an action potential. These findings provide insights into the energetic demands of EC coupling and highlight the dynamic nature of ATP concentrations in cardiac muscle. [ABSTRACT FROM AUTHOR]

CaV1.2 channels are critical players in cardiac excitation–contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation– contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility. [ABSTRACT FROM AUTHOR]

Opening the mitochondrial ATP-sensitive [K.sup.+] channel ([mitoK.sub.ATP]) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however, the mechanism of [mitoK.sub.ATP]-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the KATP channel openers diazoxide or cromakalim, their ROS production increased by 40-50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same [K.sup.+] influx as [K.sub.ATP] channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to [K.sup.+] influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated [mitoK.sub.ATP]-dependent ROS production, whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH: ubiquinone oxidoreductase) of the electron transport chain. reactive oxygen species; mitochondrial ATP-sensitive potassium channel; signaling; protein kinase C

Chagasic cardiomyopathy (CCC) is one of the main causes of heart failure and sudden death in Latin America. To date, there is no available medication to prevent or reverse the onset of cardiac symptoms. CCC occurs in a scenario of disrupted calcium dynamics and enhanced oxidative stress, which combined, may favor the hyper activation of calcium/calmodulin (Ca2+/CaM)‐calcium/calmodulin‐dependent protein kinase II (CaMKII) (Ca2+/CaM‐CaMKII) pathway, which is fundamental for heart physiology and it is implicated in other cardiac diseases. Here, we evaluated the association between Ca2+/CaM‐CaMKII in the electro‐mechanical (dys)function of the heart in the early stage of chronic experimental Trypanosoma cruzi infection. We observed that in vitro and ex vivo inhibition of Ca2+/CaM‐CaMKII reversed the arrhythmic profile of isolated hearts and isolated left‐ventricles cardiomyocytes. The benefits of the limited Ca2+/CaM‐CaMKII activation to cardiomyocytes' electrical properties are partially related to the restoration of Ca2+ dynamics in a damaged cellular environment created after T. cruzi infection. Moreover, Ca2+/CaM‐CaMKII inhibition prevented the onset of arrhythmic contractions on isolated heart preparations of chagasic mice and restored the responsiveness to the increase in the left‐ventricle pre‐load. Taken together, our data provide the first experimental evidence for the potential of targeting Ca2+/CaM‐CaMKII pathway as a novel therapeutic target to treat CCC. [ABSTRACT FROM AUTHOR]

Background: Chagas disease (CD) is a neglected disease that induces heart failure and arrhythmias in approximately 30% of patients during the chronic phase of the disease. Despite major efforts to understand the cellular pathophysiology of CD there are still relevant open questions to be addressed. In the present investigation we aimed to evaluate the contribution of the Na+/Ca2+ exchanger (NCX) in the electrical remodeling of isolated cardiomyocytes from an experimental murine model of chronic CD. Methodology/Principal findings: Male C57BL/6 mice were infected with Colombian strain of Trypanosoma cruzi. Experiments were conducted in isolated left ventricular cardiomyocytes from mice 180–200 days post-infection and with age-matched controls. Whole-cell patch-clamp technique was used to measure cellular excitability and Real-time PCR for parasite detection. In current-clamp experiments, we found that action potential (AP) repolarization was prolonged in cardiomyocytes from chagasic mice paced at 0.2 and 1 Hz. After-depolarizations, both subthreshold and with spontaneous APs events, were more evident in the chronic phase of experimental CD. In voltage-clamp experiments, pause-induced spontaneous activity with the presence of diastolic transient inward current was enhanced in chagasic cardiomyocytes. AP waveform disturbances and diastolic transient inward current were largely attenuated in chagasic cardiomyocytes exposed to Ni2+ or SEA0400. Conclusions/Significance: The present study is the first to describe NCX as a cellular arrhythmogenic substrate in chagasic cardiomyocytes. Our data suggest that NCX could be relevant to further understanding of arrhythmogenesis in the chronic phase of experimental CD and blocking NCX may be a new therapeutic strategy to treat arrhythmias in this condition. Author summary: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a neglected disease that induces heart failure and arrhythmias in approximately 30% of patients during the chronic phase of the disease. There are several substrates for arrhythmias in the heart. Some of them involve changes in the electrical properties of cardiomyocytes, the working cells of the heart. In our study we evaluate the potential involvement of Na+/Ca2+ exchanger (NCX) in the arrhythmic phenotype of cardiomyocytes isolated from mice infected with Trypanosoma cruzi, between 180- and 200- days post-infection, which is considered the chronic phase of CD in this animal model. In our study we found several arrhythmogenic membrane potential oscillations during action potential measurements, in rest and using a protocol to simulate a pause after a tachycardia. Using pharmacological approach, we determine that NCX significantly contributed to the arrhythmogenic phenomena observed. Thus, in our study we demonstrate that NCX may be relevant to the cellular arrhythmogenic profile observed in cardiomyocytes during the chronic phase of experimental CD and blocking NCX may be a new therapeutic strategy to treat arrhythmias in this condition. [ABSTRACT FROM AUTHOR]

Chagas disease (CD) is caused by the parasitic protozoan T. cruzi. The progression of CD in ~30% of patients results in Chagasic Cardiomyopathy (CCM). Currently, it is known that the inflammatory system plays a significant role in the CCM. Interferon-gamma (IFN-γ) is the major cytokine involved in parasitemia control but has also been linked to CCM. The L-type calcium current (ICa,L) is crucial in the excitation/contraction coupling in cardiomyocytes. Thus, we compared ICa,L and the mechanical properties of cardiomyocytes isolated from infected wild type (WT) and IFN-γ(−/−) mice in the first stage of T. cruzi infection. Using the patch clamp technique, we demonstrated that the infection attenuated ICa,L in isolated cardiomyocytes from the right and left ventricles of WT mice at 15 days post-infection (dpi), which was not observed in the IFN-γ(−/−) cardiomyocytes. However, ICa,L was attenuated between 26 and 30 dpi in both experimental groups. Interestingly, the same profile was observed in the context of the mechanical properties of isolated cardiomyocytes from both experimental groups. Simultaneously, we tracked the mortality and MCP-1, TNF-α, IL-12, IL-6, and IL-10 serum levels in the infected groups. Importantly, the IFN-γ(−/−) and WT mice presented similar parasitemia and serum inflammatory markers at 10 dpi, indicating that the modifications in the cardiomyocyte functions observed at 15 dpi were directly associated with IFN-γ(−/−) deficiency. Thus, we showed that IFN-γ plays a crucial role in the electromechanical remodeling of cardiomyocytes during experimental T. cruzi infection in mice. [ABSTRACT FROM AUTHOR]

Perfusion of the heart with bradykinin triggers cellular signaling events that ultimately cause opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels, increased H2O2 production, inhibition of the mitochondrial permeability transition (MPT), and cardioprotection. We hypothesized that the interaction of bradykinin with its receptor induces the assembly of a caveolar signaling platform (signalosome) that contains the enzymes of the signaling pathway and that migrates to mitochondria to induce mitoKATP channel opening. We developed a novel method for isolating and purifying signalosomes from Langendorff-perfused rat hearts treated with bradykinin. Fractions containing the signalosomes were found to open mitOKATP channels in mitochondria isolated from untreated hearts via the activation of mitochondrial PKC-ε. mitoKATP channel opening required signalosome-dependent phosphorylation of an outer membrane protein. Immunodetection analysis revealed the presence of the bradykinin B2 receptor only in the fraction isolated from bradykinin-treated hearts. Immunodetection and immunogold labeling of caveolin-3, as well as sensitivity to cholesterol depletion and resistance to Triton X-100, attested to the caveolar nature of the signalosomes. Ischemic preconditioning, ischemic postconditioning, and perfusion with ouabain also led to active signalosome fractions that opened mitoKATP channels in mitochondria from untreated hearts. These results provide initial support for a novel mechanism for signal transmission from a plasma membrane receptor to mitoKATP channels. [ABSTRACT FROM AUTHOR]

Activation of protein ki- nase Ce (PKCe), opening of mitochondrial AlP-sensitive K~ chan- nels (mitoKA-rp), and increased mitochondrial reactive oxygen specie~ (ROS) are key events in the signaling that underlies cardioprotection We showed previously that mitoKATP is opened by activation of mitochondrial PKCe, designated PKCeI, that is closely associatec with mitoKATP. mitoKA-rp opening then causes an increase in ROS production by complex I of the respiratory chain. This ROS activates a second pool of PKCe, designated PKCe2, which inhibits the mito- chondrial permeability transition (MPT). In the present study, wc measured mitoKATP-dependent changes in mitochondrial matrix vol- ume to further investigate the relationships among PKCe, mitoKATP, ROS, and MPT. We present evidence that I) mitoKATP can be openec by H202 and nitric oxide (NO) and that these effects are mediated b) PKCcI and not by direct actions on mitoKATP, 2) superoxide has nc effect on mitoKATP opening, 3) exogenous H2O2 or NO also inhibit~ MPT opening, and both compounds do so independently of mitoKA-rF activity via activation of PKCe2, 4) mitoKA-rP opening induced b~, PKG, phorbol ester, or diazoxide is not mediated by ROS, anc 5) mitoKATP-generated ROS activates PKCeI and induces phospho- rylation-dependent mitoKATP opening in vitro and in vivo. Thia mitoKATP-dependent mitoKATP opening constitutes a positive feed- back loop capable of maintaining the channel open after the stimulia is no longer present. This feedback pathway may be responsible foi the lasting protective effect of preconditioning, colloquially known a~ the memory effect. [ABSTRACT FROM AUTHOR]

Much of cell death from ischaemia/reperfusion in heart and other tissues is generally thought to arise from mitochondrial permeability transition (MPT) in the first minutes of reperfusion. In ischaemic pre-conditioning, agonist binding to Gi protein-coupled receptors prior to ischaemia triggers a signalling cascade that protects the heart from MPT. We believe that the cytosolic component of this trigger pathway terminates in activation of guanylyl cyclase resulting in increased production of cGMP and subsequent activation of protein kinase G (PKG). PKG phosphorylates a protein on the mitochondrial outer membrane (MOM), which then causes the mitochondrial KATP channel (mitoKATP) on the mitochondrial inner membrane to open, leading to increased production of reactive oxygen species (ROS) by the mitochondria. This implies that the protective signal is somehow transmitted from the MOM to its inner membrane. This is accomplished by a series of intermembrane signalling steps that includes protein kinase C (PKCɛ) activation. The resulting ROS then activate a second PKC pool which, through another signal transduction pathway termed the mediator pathway, causes inhibition of MPT and reduction in cell death. [ABSTRACT FROM PUBLISHER]

We showed recently that mitochondrial ATP-dependent K+ channel (mitoKATP) opening is required for the inotropic response to ouabain. Because mitoKATP opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na+-K+-ATPase with the opening of mitoKATP. In Langendorff-perfused rat hearts, 10-80 µM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoKATP opening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoKATP inhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection. [ABSTRACT FROM AUTHOR]

Opening the mitochondrial ATP-sensitive K+ channel (mitoKATP) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however, the mechanism of mitoKATP-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the KATP channel openers diazoxide or cromakalim, their ROS production increased by 40-50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same K+ influx as KATP channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to K+ influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated mitoKATP-dependent ROS production, whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH: ubiquinone oxidoreductase) of the electron transport chain. [ABSTRACT FROM AUTHOR]

Myocardial infarction is a manifestation of necrotic cell death as a result of opening of the mitochondrial permeability transition (MPT). Receptor-mediated cardioprotection is triggered by an intracellular signaling pathway that includes phosphatidylinositol 3-kinase, endothelial nitric-oxide synthase, guanylyl cyclase, protein kinase G (PKG), and the mitochondrial KATP channel (mitoKATP). In this study, we explored the pathway that links mitoKATP with the MPT. We confirmed previous findings that diazoxide and activators of PKG or protein kinase C (PKC) inhibited MPT opening. We extended these results and showed that other K+ channel openers as well as the K+ ionophore valinomycin also inhibited MPT opening and that this inhibition required reactive oxygen species. By using isoform-specific peptides, we found that the effects of KATP channel openers, PKG, or valinomycin were mediated by a PKC∊. Activation of PKC∊ by phorbol 12-myristate 13-acetate or H2O2 resulted in mitoKATP-independent inhibition of MPT opening, whereas activation of PKC∊ by PKG or the specific PKC∊ agonist ψ∊ receptor for activated C kinase caused mitoKATP-dependent inhibition of MPT opening. Exogenous H2O2 inhibited MPT, because of its activation of PKC∊, with an IC50 of 0.6 (±0.1) μ. On the basis of these results, we propose that two different PKC∊ pools regulate this signaling pathway, one in association with mitoKATP and the other in association with MPT. [ABSTRACT FROM AUTHOR]

This study investigates the role of the mitochondrial ATP-sensitive K+ channel (mitoKATP) in response to positive inotropic stress. In Langendorff-perfused rat hearts, inotropy was induced by increasing perfusate calcium to 4 mM, by adding 80 μM ouabain or 0.25 μM dobutamine. Each of these treatments resulted in a sustained increase in rate-pressure product (RPP) of ∼60%. Inhibition of mitoKATP by perfusion of 5-hydroxydecanoate (5-HD) or tetraphenylphosphonium before induction of inotropic stress resulted in a marked attenuation of RPP. Inhibition of mitoKATP after induction of stress caused the inability of the heart to maintain a high-work state. In human atrial fibers, the increase in contractility induced by dobutamine was inhibited 60% by 5-HD. In permeabilized fibers from the Langendorff-perfused rat hearts, inhibition of mitoKATP resulted, in all cases, in an alteration of adenine nucleotide compartmentation, as reflected by a 60% decrease in the half-saturation constant for ADP [K1/2 (ADP)]. We conclude that opening of cardiac mitoKATP is essential for an appropriate response to positive inotropic stress and propose that its involvement proceeds through the prevention of stress-induced decrease in mitochondrial matrix volume. These results indicate a physiological role for mitoKATP, in inotropy and, by extension, in heart failure. [ABSTRACT FROM AUTHOR]

The mitochondrial ATP-sensitive K+ channel (mitoKATP) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoKATP opening that should be observable at the level of the mitochondrion. MitoKATP opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoKATP opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoKATP openers and blockers with those of valinomycin, we show that four independent assays of mitoKATP activity yield quantitatively identical results for mitoKATP-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoKATP opening for mitochondria. [ABSTRACT FROM AUTHOR]

The author examines a study on myocardial regeneration and functional recovery following cell activation in hearts with injuries. He points out that cell cycle activation is incapable of promoting full recovery of damaged hearts because of the progression of cell division through restriction check points. He argues that using constitutively active promoters that target cell proliferation-related genes is not appropriate for damaged hearts.

The ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoKATP) is a highly selective conductor of K+ ions. When isolated in the presence of nonionic detergent and reconstituted in liposomes, mitoKATP is inhibited with high affinity by ATP (K½ = 20-30 µM). We have suggested that holo-mitoKATP is a heteromultimer consisting of an inwardly rectifying K+ channel (mitoKIR) and a sulfonylurea receptor (Grover, G. J., and Garlid, K. D. (2000) J. Mol. Cell Cardiol. 32, 677-695). Here, we show that a 55-kDa protein isolated by ethanol extraction and reconstituted in bilayer lipid membranes and liposomes is the mitoKIR. This protein, which lacks the sulfonylurea receptor subunit, is inhibited with low affinity by ATP, with K½ ∼ 550 µM. ATP inhibition of both mitoKIR and holo-mitoKATP is reversed by UDP (K½ = 10-15 µM). Holo-mitoKATP is opened by cromakalim and diazoxide, and the flux through the open channel is inhibited by glibenclamide and 5-hydroxydecanoate. None of these agents has any effect upon mitoKIR. We have identified two compounds that act specifically on mitoKIR, p-diethylaminoethylbenzoate reverses inhibition of mitoKIR by ATP and ADP at micromolar concentrations and also opens mitoKATP in isolated mitochondria. Tetraphenylphosphonium inhibits K+ flux through both mitoKIR and mitoKATP with the same apparent affinity. These findings support the hypothesis that the 55-kDa mitoKIR is the channel component of mitoKATP. [ABSTRACT FROM AUTHOR]

The L-type Ca 2+ channel (Ca V 1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca 2+ flux that drives Ca 2+ -induced-Ca 2+ -release, Ca V 1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune Ca V 1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca 2+ channel trafficking in non-myocytes, however whether 14-3-3 has similar effects on Ca V 1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including Na V 1.5 and hERG. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with Ca V 1.2 in a phosphorylation-dependent manner and regulates cardiac Ca V 1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, super-resolution imaging, and co-immunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with Ca V 1.2. The degree of 14-3-3/Ca V 1.2 colocalization increased upon stimulation of β -adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated Ca V 1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs Ca V 1.2 super-clustering. 14-3-3 overexpression increased basal Ca V 1.2 cluster size and Ca 2+ currents in ventricular myocytes, with maintained channel responsivity to isoproterenol. In contrast, isoproterenol-stimulated augmentation of sarcolemmal Ca V 1.2 expression and currents in ventricular myocytes were abrogated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with Ca V 1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β -adrenergic stimulation., Significance Statement: The L-type Ca 2+ channel, Ca V 1.2, plays an essential role in excitation-contraction coupling in the heart and in part regulates the overall strength of contraction during basal and fight- or-flight β -adrenergic signaling conditions. Proteins that modulate the trafficking and/or activity of Ca V 1.2 are interesting both from a physiological and pathological perspective, since alterations in Ca V 1.2 can impact action potential duration and cause arrythmias. A small protein called 14-3-3 regulates other ion channels in the heart and other Ca 2+ channels, but how it may interact with Ca V 1.2 in the heart has never been studied. Examining factors that affect Ca V 1.2 at rest and during β -adrenergic stimulation is crucial for our ability to understand and treat disease and aging conditions where these pathways are altered.

Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2-8 °C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with Cyclospora cayetanensis oocysts, as well as açai pulp and sugarcane juice tainted with Trypanosoma cruzi trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions. (170 words)., Competing Interests: Instituto de Biologia Molecular do Paraná (IBMP) produces both qPCR mastermixes used in the present study. BNDES or IBMP had no participation in the study's design, data collection, analysis, interpretation, or writing of the report, and decision to submit for publication. The authors declare no further competing interests., (© 2021 The Authors.)

Although molecular diagnostics is well established in clinical laboratories, its full potential has not been extended to field settings. Typically, diagnostic real-time quantitative PCR (qPCR) reagents require temperature-controlled transportation and storage. Furthermore, thermocyclers are bulky and fragile, requiring good infrastructure for optimal operation. These major hurdles strongly limit use of molecular-based tests in low-resource scenarios. Herein, Trypanosoma cruzi or Plasmodium spp. DNA were detected with qPCR using commercial equipment (ABI7500 instrument) and a prototype platform comprising a portable device and a silicon chip, named Q3-Plus. In addition, a ready-to-use reaction format, where all qPCR reagents are stored on plate or on chip, was compared with the traditional freezer-stored format. No significant differences were observed in detecting T. cruzi or Plasmodium spp. DNA between thermocyclers, as well as between reagents' formats, for storage periods of up to 28 days (at 2°C to 8°C or 21°C to 23°C, respectively). When challenged with patients' samples, the Q3-Plus system performed as efficiently as the standard equipment for Plasmodium spp. DNA detection, showing it to be a valuable solution to malaria point-of-care diagnostics. Detection of T. cruzi DNA in chronic patients' samples using the Q3-Plus system yielded approximately 50% efficiency relative to the ABI7500. These results are essential to support future endeavors to bring molecular diagnostics to the point of care, where most needed., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Mammalian mitochondria, as well as rat, plant and Caenorhabditis elegans mitochondria, possess an ATP-sensitive K+ channel (mitoK(ATP)) that has been pharmacologically characterised. Opening of mitoK(ATP) and the subsequent K+ entry into the matrix was shown to have three effects on mitochondria physiology: (i) an increase in matrix volume (swelling), (ii) an acceleration of respiration, and (iii) an increase in reactive oxygen species (ROS) production. These effects on mitochondria bioenergetics have been shown to be part of distinct intracellular signalling pathways, to protect against cell death and to modulate gene transcription. To date, such a channel or its activity has not been described in trypanosomatids. In the present study, we show pharmacological evidence for the presence of a mitoK(ATP) in trypanosomatids. Cells were incubated in a hypotonic medium followed by mild detergent exposure to isolate mitoplasts from Trypanosoma cruzi and Crithidia fasciculata. Mitoplasts swelled when incubated in KCl medium due to respiration-driven K+ entry into the matrix. Swelling was sensitive to the presence of ATP when the mitoplast suspension was incubated in K+ -containing, but not in K+ -free, medium. The ATP inhibition of swelling was reversed by the mitoK(ATP) agonist diazoxide and the diazoxide-induced swelling was inhibited by the mitoK(ATP) blockers 5-hydroxydecanoate (5HD) or glibenclamide. Similar to mammalian and rat mitochondria, trypanosomatid mitoK(ATP) activity was modulated by the general protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA) and antagonist chelerythrine. As expected, the potassium ionophore valinomycin could also reverse the ATP-inhibited state but this reversal was not sensitive to 5HD or glibenclamide. Dose response curves for ATP, diazoxide and 5HD are presented. These results provide strong evidence for the presence of an ATP-sensitive K+ in trypanosomatid mitochondria.

Mitochondria are central players in the pathophysiology of ischemia-reperfusion. Activation of plasma membrane G-coupled receptors or the Na,K-ATPase triggers cytosolic signaling pathways that result in cardioprotection. Our working hypothesis is that the occupied receptors migrate to caveolae, where signaling enzymes are scaffolded into signalosomes that bud off the plasma membrane and migrate to mitochondria. The signalosome-mitochondria interaction then initiates intramitochondrial signaling by opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). MitoK(ATP) opening causes an increase in ROS production, which activates mitochondrial protein kinase C epsilon (PKCvarepsilon), which inhibits the mitochondrial permeability transition (MPT), thus decreasing cell death. We review the experimental findings that bear on these hypotheses and other modes of protection involving mitochondria.

Background: Mitochondrial (m) adenosine triphosphate sensitive potassium (K(ATP)) channel opening has been reported to trigger and/or mediate cardioprotection by volatile anesthetics. However, the effects of volatile anesthetics on mitochondrial function are not well understood. Prevention of mitochondrial matrix volume (MMV) contraction during ischemia may contribute to cardioprotection against ischemia/reperfusion injury. We investigated whether sevoflurane increases MMV and if this increase is mediated by mK(ATP) channel opening., Methods: Mitochondria from fresh guinea pig hearts were isolated and diluted in buffer that included oligomycin and ATP to inhibit ATP synthesis. Changes in MMV by diazoxide, a known mK(ATP) channel opener, and by different sevoflurane concentrations, were measured by light absorption at 520 nm in the absence or presence of the mK(ATP) channel blocker, 5-hydroxydecanoate., Results: Compared with control, 30-300 microM sevoflurane (approximately 0.2-2.1 vol %) increased MMV by 30%-55%, which was similar to the effect of diazoxide. These increases were blocked by 5-hydroxydecanoate. Higher sevoflurane concentration (1000 microM; 7.1 vol %), however, had no effect on MMV., Conclusions: In clinically relevant concentrations, sevoflurane increases MMV via mK(ATP) channel opening. Preservation of mitochondrial integrity may contribute to the cardioprotective effects of sevoflurane against ischemia/reperfusion injury. Impaired mitochondrial function at supraclinical anesthetic concentrations may explain the observed biphasic response. These findings add to our understanding of the intracellular mechanisms of volatile anesthetics as cardioprotective drugs.

The aim of this study was to investigate the effects of HMR1098, a selective blocker of sarcolemmal ATP-sensitive potassium channel (sarcK(ATP)), in Langendorff-perfused rat hearts submitted to ischemia and reperfusion. The recovery of heart hemodynamic and mitochondrial function, studied on skinned fibers, was analyzed after 30-min global ischemia followed by 20-min reperfusion. Infarct size was quantified on a regional ischemia model after 2-h reperfusion. We report that the perfusion of 10 microM HMR1098 before ischemia, delays the onset of ischemic contracture, improves recovery of cardiac function upon reperfusion, preserves the mitochondrial architecture, and finally decreases infarct size. This HMR1098-induced cardioprotection is prevented by 1 mM 2-mercaptopropionylglycine, an antioxidant, and by 100 nM nifedipine, an L-type calcium channel blocker. Concomitantly, it is shown that HMR1098 perfusion induces (i) a transient and specific inhibition of the respiratory chain complex I and, (ii) an increase in the averaged intracellular calcium concentration probed by the in situ measurement of indo-1 fluorescence. Finally, all the beneficial effects of HMR1098 were strongly inhibited by 5-hydroxydecanoate and abolished by glibenclamide, two mitoK(ATP) blockers. This study demonstrates that the HMR1098-induced cardioprotection occurs indirectly through extracellular calcium influx, respiratory chain complex inhibition, reactive oxygen species production and mitoK(ATP) opening. Taken together, these data suggest that a functional interaction between sarcK(ATP) and mitoK(ATP) exists in isolated rat heart ischemia model, which is mediated by extracellular calcium influx.

Mitochondria are key mediators of the cardioprotective signal and the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) plays a crucial role in originating and transmitting that signal. Recently, protein kinase C epsilon (PKC epsilon) has been identified as a component of the mitoK(ATP) signaling cascade. We hypothesized that PKC epsilon and mitoK(ATP) interact directly to form functional signaling modules in the inner mitochondria membrane. To examine this possibility, we studied K+ flux in liposomes containing partially purified mitoK(ATP). The reconstituted proteins were obtained after detergent extraction of isolated mitochondria, 200-fold purification by ion exchange chromatography, and reconstitution into lipid vesicles. Immunoblot analysis revealed the presence of PKC epsilon in the reconstitutively active fraction. Addition of the PKC activators 12-phorbol 13-myristate acetate, hydrogen peroxide, and the specific PKC epsilon peptide agonist, psi epsilonRACK, each activated mitoK(ATP)-dependent K+ flux in the reconstituted system. This effect of PKC epsilon was prevented by chelerythrine, by the specific PKC epsilon peptide antagonist, epsilonV(1-2), and by the specific mitoK(ATP) inhibitor 5-hydroxydecanoate. In addition, the activating effect of PKC agonists was reversed by exogenous protein phosphatase 2A. These results demonstrate persistent, functional association of mitochondrial PKC epsilon and mitoK(ATP).

Bepridil, which is clinically useful in the treatment of arrhythmias, has been reported to inhibit sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels. However, the effect of bepridil on mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels remains unclear. The objective of the present study was to determine whether bepridil activates mitoK(ATP) channels and confers cardioprotection. SarcK(ATP) channels composed of Kir6.2+SUR2A in human embryonic kidney (HEK) 293 cells were examined using the patch-clamp technique. Flavoprotein fluorescence in guinea pig ventricular cells and matrix volume in isolated rat heart mitochondria were measured to assay mitoK(ATP) channel activity. Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) was measured by loading cells with rhod-2 fluorescence. Coronary-perfused guinea pig ventricular muscles were subjected to 35-min no-flow ischemia followed by 60-min reperfusion. Bepridil (10 microM) completely inhibited the pinacidil-induced Kir6.2+SUR2A channel current expressed in HEK 293 cells. Bepridil reversibly oxidized the flavoprotein and increased mitochondrial matrix volume in a concentration-dependent manner. Furthermore, bepridil significantly attenuated the ouabain-induced increase of [Ca(2+)](m). Pretreatment with bepridil for 5 min before ischemia improved the recovery of developed tension measured after 60 min of reperfusion. These effects of bepridil were abolished by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) and by the nonselective K(ATP) channel blocker glisoxepide (10 microM). Our results indicate that bepridil is an opener of mitoK(ATP) channels but an inhibitor of sarcK(ATP) channels and exerts a direct cardioprotective effect on native cardiac myocytes. This is the first report of a unique modulator of K(ATP) channels; bepridil would be expected to mitigate ischemic injury while blunting arrhythmias.

Ischemic and pharmacological preconditioning can be triggered by an intracellular signaling pathway in which Gi-coupled surface receptors activate a cascade including phosphatidylinositol 3-kinase, endothelial nitric oxide synthase, guanylyl cyclase, and protein kinase G (PKG). Activated PKG opens mitochondrial KATP channels (mitoKATP) which increase production of reactive oxygen species. Steps between PKG and mitoKATP opening are unknown. We describe effects of adding purified PKG and cGMP on K+ transport in isolated mitochondria. Light scattering and respiration measurements indicate PKG induces opening of mitoKATP similar to KATP channel openers like diazoxide and cromakalim in heart, liver, and brain mitochondria. This effect was blocked by mitoKATP inhibitors 5-hydroxydecanoate, tetraphenylphosphonium, and glibenclamide, PKG-selective inhibitor KT5823, and protein kinase C (PKC) inhibitors chelerythrine, Ro318220, and PKC-epsilon peptide antagonist epsilonV(1-2). MitoKATP are opened by the PKC activator 12-phorbol 13-myristate acetate. We conclude PKG is the terminal cytosolic component of the trigger pathway; it transmits the cardioprotective signal from cytosol to inner mitochondrial membrane by a pathway that includes PKC-epsilon.

Coronary artery disease and its sequelae-ischemia, myocardial infarction, and heart failure-are leading causes of morbidity and mortality in man. Considerable effort has been devoted toward improving functional recovery and reducing the extent of infarction after ischemic episodes. As a step in this direction, it was found that the heart was significantly protected against ischemia-reperfusion injury if it was first preconditioned by brief ischemia or by administering a potassium channel opener. Both of these preconditioning strategies were found to require opening of a K(ATP) channel, and in 1997 we showed that this pivotal role was mediated by the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). This paper will review the evidence showing that opening mitoK(ATP) is cardioprotective against ischemia-reperfusion injury and, moreover, that mitoK(ATP) plays this role during all three phases of the natural history of ischemia-reperfusion injury preconditioning, ischemia, and reperfusion. We discuss two distinct mechanisms by which mitoK(ATP) opening protects the heart-increased mitochondrial production of reactive oxygen species (ROS) during the preconditioning phase and regulation of intermembrane space (IMS) volume during the ischemic and reperfusion phases. It is likely that cardioprotection by ischemic preconditioning (IPC) and K(ATP) channel openers (KCOs) arises from utilization of normal physiological processes. Accordingly, we summarize the results of new studies that focus on the role of mitoK(ATP) in normal cardiomyocyte physiology. Here, we observe the same two mechanisms at work. In low-energy states, mitoK(ATP) opening triggers increased mitochondrial ROS production, thereby amplifying a cell signaling pathway leading to gene transcription and cell growth. In high-energy states, mitoK(ATP) opening prevents the matrix contraction that would otherwise occur during high rates of electron transport. MitoK(ATP)-mediated volume regulation, in turn, prevents disruption of the structure-function of the IMS and facilitates efficient energy transfers between mitochondria and myofibrillar ATPases.