Loss Of Mitochondrial Function Research Articles

Isolation, characterization and mechanism of action of an antimicrobial peptide from Lecythis pisonis seeds with inhibitory activity against Candida albicans.

Antimicrobial peptides (AMPs) are produced by a range of organisms as a first line of defense against invaders or competitors. Owing to their broad antimicrobial activity, AMPs have attracted attention as a potential source of chemotherapeutic drugs. The increasing prevalence of infections caused by Candida species as opportunistic pathogens in immunocompromised patients requires new drugs. Lecythis pisonis is a Lecythydaceae tree that grows in Brazil. The AMPs produced by this tree have not been described previously. We describe the isolation of 12 fractions enriched in peptides from L. pisonis seeds. Of the 12 fractions, at 10 μg/ml, the F4 fraction had the strongest growth inhibitory effect (53.7%) in Candida albicans, in addition to a loss of viability of 94.9%. The F4 fraction was separated into seven sub-fractions by reversed-phase chromatography. The F4.7' fraction had the strongest activity at 10 μg/ml, inhibiting C. albicans growth by 38.5% and a 69.3% loss of viability. The peptide in F4.7' was sequenced and was found to be similar to plant defensins. For this reason, the peptide was named L. pisonis defensin 1 (Lp-Def1). The mechanism of action that is responsible for C. albicans inhibition by Lp-Def1 includes a slight increase of reactive oxygen species induction and a significant loss of mitochondrial function. The results described here support the future development of plant defensins, specifically Lp-Def1, as new therapeutic substances against fungi, especially C. albicans.

Extracellular polymeric substance from Aphanizomenon flos-aquae induces apoptosis via the mitochondrial pathway in A431 human epidermoid carcinoma cells.

Extracellular polymeric substance (EPS) is a substance secreted during algal growth, which has been found to have numerous health-promoting effects. In the present study, A431 human epidermoid carcinoma cells were selected as target cells and cultivated in vitro as an experimental model to investigate the anti-cancer effect of extracellular polymeric substances from Aphanizomenon flos-aquae (EPS-A) and the possible underlying mechanism. Apoptosis- and cell cycle-associated molecules as well as the mitochondrial membrane potential of the cells were quantified using flow cytometry (FCM). FCM showed that EPS-A induced cell cycle arrest, which led to a loss of mitochondrial function of the A431 cells and an increase in necrotic and late apoptotic cells. In order to evaluate the apoptosis and cell viability, acridine orange/ethidium bromide staining was used, morphological changes were observed using fluorescence microscopy and typical apoptotic characteristics were observed. Following treatment with a high dose of EPS-A, transmission electron microscopy showed nuclear fragmentation, chromosome condensation, cell shrinkage and expansion of the endoplasmic reticulum; apoptotic bodies were also observed. In conclusion, EPS-A caused cell cycle arrest, stimulated cell apoptosis via the mitochondrial pathway and exhibited important anti-cancer activity.

Exercise training normalizes mitochondrial respiratory capacity within the striatum of the R6/1 model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive cell loss in the striatum and cerebral cortex, leading to a decline in motor control and eventually death. The mechanisms promoting motor dysfunction are not known, however loss of mitochondrial function and content has been observed, suggesting that mitochondrial dysfunction may contribute to HD phenotype. Recent work has demonstrated that voluntary wheel running reduces hindlimb clasping in the R6/1 mouse model of HD, which we hypothesized may be due to preservation of mitochondrial content with exercise. Therefore, we investigated the role of chronic exercise training on preventing symptom progression and the loss of mitochondrial content in HD. Exercising R6/1 mice began training at 7 wks of age and continued for 10 or 20 wks. At 17 wks of age, R6/1 mice displayed a clasping phenotype without showing changes in mitochondrial respiration or protein content in either the cortex or striatum, suggesting mitochondrial dysfunction is not necessary for the progression of symptoms. At 27 wks of age, R6/1 mice demonstrated no additional changes in mitochondrial content or respiration within the cortex, but displayed loss of protein in complexes I and III of the striatum, which was not present in exercise-trained R6/1 mice. Mitochondrial respiration was also elevated in the striatum of R6/1 mice at 27 wks, which was prevented with exercise training. Together, the present study provides evidence that mitochondrial dysfunction is not necessary for the progression of hindlimb clasping in R6/1 mice, and that exercise partially prevents changes in mitochondrial content and function that occur late in HD.

Eicosapentaenoic acid but not docosahexaenoic acid restores skeletal muscle mitochondrial oxidative capacity in old mice.

Mitochondrial dysfunction is often observed in aging skeletal muscle and is implicated in age-related declines in physical function. Early evidence suggests that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) improve mitochondrial function. Here, we show that 10 weeks of dietary eicosapentaenoic acid (EPA) supplementation partially attenuated the age-related decline in mitochondrial function in mice, but this effect was not observed with docosahexaenoic acid (DHA). The improvement in mitochondrial function with EPA occurred in the absence of any changes in mitochondrial abundance or biogenesis, which was evaluated from RNA sequencing, large-scale proteomics, and direct measurements of muscle mitochondrial protein synthesis rates. We find that EPA improves muscle protein quality, specifically by decreasing mitochondrial protein carbamylation, a post-translational modification that is driven by inflammation. These results demonstrate that EPA attenuated the age-related loss of mitochondrial function and improved mitochondrial protein quality through a mechanism that is likely linked with anti-inflammatory properties of n-3 PUFAs. Furthermore, we demonstrate that EPA and DHA exert some common biological effects (anticoagulation, anti-inflammatory, reduced FXR/RXR activation), but also exhibit many distinct biological effects, a finding that underscores the importance of evaluating the therapeutic potential of individual n-3 PUFAs.

Mitochondrial function and lifespan of mice with controlled ubiquinone biosynthesis.

Ubiquinone (UQ) is implicated in mitochondrial electron transport, superoxide generation and as a membrane antioxidant. Here we present a mouse model in which UQ biosynthesis can be interrupted and partially restored at will. Global loss of UQ leads to gradual loss of mitochondrial function, gradual development of disease phenotypes and shortened lifespan. However, we find that UQ does not act as antioxidant in vivo and that its requirement for electron transport is much lower than anticipated, even in vital mitochondria-rich organs. In fact, severely depressed mitochondrial function due to UQ depletion in the heart does not acutely impair organ function. In addition, we demonstrate that severe disease phenotypes and shortened lifespan are reversible upon partial restoration of UQ levels and mitochondrial function. This observation strongly suggests that the irreversible degenerative phenotypes that characterize ageing are not secondarily caused by the gradual mitochondrial dysfunction that is observed in aged animals.

Novel antioxidants protect mitochondria from the effects of oligomeric amyloid beta and contribute to the maintenance of epigenome function.

Alzheimer's disease is associated with metabolic deficits and reduced mitochondrial function, with the latter due to the effects of oligomeric amyloid beta peptide (AβO) on the respiratory chain. Recent evidence has demonstrated reduction of epigenetic markers, such as DNA methylation, in Alzheimer's disease. Here we demonstrate a link between metabolic and epigenetic deficits via reduction of mitochondrial function which alters the expression of mediators of epigenetic modifications. AβO-induced loss of mitochondrial function in differentiated neuronal cells was reversed using two novel antioxidants (1 and 2); both have been shown to mitigate the effects of reactive oxygen species (ROS), and compound 1 also restores adenosine triphosphate (ATP) levels. While both compounds were effective in reducing ROS, restoration of ATP levels was associated with a more robust response to AβO treatment. Our in vitro system recapitulates key aspects of data from Alzheimer's brain samples, the expression of epigenetic genes in which are also shown to be normalized by the novel analogues.

Role of mitochondria in regulating microRNA activity and its relevance to the central nervous system

Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. Aberrant mitochondria function has been linked to diverse and complex human diseases such as neurodegenerative diseases, cancers, myopathies, premature aging, and metabolic syndromes (Nunnari and Suomalainen, 2012). For example, there is clear evidence of a rapid and sustained loss of mitochondrial function following traumatic injury to the central nervous system (CNS) (Sullivan et al., 2005; McEwen et al., 2011). However, our understanding of how compromised mitochondrial activity impacts overall cellular function in the CNS is far from complete.

Cytotoxic effect of carotenoid phytonutrient lycopene on P. falciparum infected erythrocytes

Resistance to almost all class of antimalarial drugs has emerged as one of the greatest challenges to malaria control. Strategies to limit the advent and spread of drug-resistant malaria include development of new drugs and its combination with existing drugs. In this work we provide a strong evidence for phytonutrient lycopene, a non-provitamin A carotenoid, to be effective against Plasmodium falciparum growth in vitro. Consistent with the previous findings in mammalian cells, lycopene's prooxidant activity promoted the production of reactive oxygen species (ROS) in P. falciparum. Also a significant loss of mitochondrial functionality and thus, the loss of the membrane potential was observed in lycopene treated schizonts. Taken together, our results indicated that the generation of ROS and loss of mitochondrial membrane potential accounted for lycopene's cytotoxicity against P. falciparum growth in vitro. These insights will help in the design of new treatment strategies to combat malaria.

Mercury-induced dysfunctions in multiple organelles leading to cell death

Mercury (Hg) is a highly toxic metal that can exert multiple adverse effects, ultimately leading to cell death. Before causing death, the Hg enters the cells and affects diverse intracellular targets. The present study aimed to investigate the structure and function of several organelles or cellular structures, including mitochondria, acidic compartments and vesicles, endoplasmic reticulum elements and microfilaments, following Hg exposure of a human hepatic cell line (HuH-7 cells) to examine the sequence and coordination of the events associated with Hg-induced cell death. Hg exposure led to a progressive decrease in cell viability and induced alterations in cell morphology including cytoplasmic shrinkage and nuclear fragmentation. Hg treatment (10μM for 12h) affected multiple intracellular targets simultaneously. These included loss of mitochondrial functionality, pronounced cytoplasmic acidification and dysfunctions in the cytoskeleton and endoplasmic reticulum. This overall Hg-induced toxicity in the human hepatocyte cell line (HuH-7 cells) led to cell death through both apoptosis and autophagy.

Involvement of heme oxygenase-1 in neuroprotection by sanguinarine against glutamate-triggered apoptosis in HT22 neuronal cells

Sanguinarine is a natural compound isolated from the roots of Macleaya cordata and M. microcarpa, has been reported to possess several biological activities such as anti-inflammatory and anti-oxidant effects. In the present study, we demonstrated that sanguinarine markedly induces the expression of HO-1 which leads to a neuroprotective response in mouse hippocampus-derived neuronal HT22 cells from apoptotic cell death induced by glutamate. Sanguinarine significantly attenuated the loss of mitochondrial function and membrane integrity associated with glutamate-induced neurotoxicity. Sanguinarine protected against glutamate-induced neurotoxicity through inhibition of HT22 cell apoptosis. JC-1 staining, which is a well-established measure of mitochondrial damage, was decreased after treatment with sanguinarine in glutamate-challenged HT22cells. In addition, sanguinarine diminished the intracellular accumulation of ROS and Ca2+. Sanguinarine also induced HO-1, NQO-1 expression via activation of Nrf2. Additionally, we found that si RNA mediated knock-down of Nrf2 or HO-1 significantly inhibited sanguinarine-induced neuroprotective response. These findings revealed the therapeutic potential of sanguinarine in preventing the neurodegenerative diseases.

Paradoxical cellular effects and biological role of the multifaceted compound nordihydroguaiaretic acid.

Nordihydroguaiaretic acid (NDGA) is a phenolic compound obtained from the leaves of the evergreen desert shrub Larrea tridentata (Creosote bush), which has been used anciently in folk medicine for the treatment of multiple diseases. At the molecular level, NDGA is a potent scavenger of reactive oxygen species. Lipoxygenase inhibition by NDGA has been broadly studied over several cell models; however, NDGA exerts other antioxidant properties and cytoprotective effects in non-tumor cells, which are related with its role as modulator of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) antioxidant pathway. In contrast, in tumor cells NDGA exerts pro-apoptotic activity and anti-tumor effects. Different effects of NDGA have been observed in mitochondria, where NDGA prevents mitochondrial damage in non-tumor cells and induces loss of mitochondrial function in tumor cells. Moreover, NDGA exerts beneficial effects in diverse diseases like cancer, renal damage, Huntington's disease, Alzheimer's disease, and other neurodegenerative pathologies. This work represents a critical review about relevant NDGA mechanisms, cellular effects, and signal pathways involved with possible useful effects.

Involvement of activation of the Nrf2/ARE pathway in protection against 6-OHDA-induced SH-SY5Y cell death by α-iso-cubebenol

Free radical-mediated neurodegeneration is one of the many causes of Parkinson's disease (PD). As part of our ongoing studies on the identification of biologically active Schisandra chinensis components, we have isolated and structurally elucidated α-iso-cubebenol. This study was carried out in an attempt to clarify the neuroprotective effect of α-iso-cubebenol on toxin-insulted dopaminergic neuronal death using 6-hydroxy-dopamine (6-OHDA)-induced dopaminergic SH-SY5Y cells. α-iso-cubebenol significantly attenuated the loss of mitochondrial function (MTT assay) and membrane integrity (lactate dehydrogenase assay) associated with 6-OHDA-induced neurotoxicity. Pretreatment of the cells with α-iso-cubebenol diminished the intracellular accumulation of reactive oxygen species (ROS) and calcium in response to 6-OHDA. Moreover, α-iso-cubebenol protected against 6-OHDA-induced neurotoxicity through inhibition of SH-SY5Y cell apoptosis. In addition, JC-1 staining, which is a well-established measure of mitochondrial damage, was decreased after treatment with α-iso-cubebenol. Notably, α-iso-cubebenol inhibited the release of mitochondrial flavoprotein apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus following 6-OHDA treatment. In addition, α-iso-cubebenol reduced the 6-OHDA-induced phosphorylation of ERK and induced the phosphorylation of PKA, PKB, and CREB in a dose-dependent manner. Moreover, α-iso-cubebenol stimulated the activation of Nrf2, a downstream target of CREB. Furthermore, α-iso-cubebenol stimulated the expression of multiple antioxidant response genes (NQO-1 and HO-1). Finally, CREB and Nrf2 siRNA transfection diminished α-iso-cubebenol-mediated neuroprotection.

Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line.

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Sexual Dimorphism in the Alterations of Cardiac Muscle Mitochondrial Bioenergetics Associated to the Ageing Process

The incidence of cardiac disease is age and sex dependent, but the mechanisms governing these associations remain poorly understood. Mitochondria are the organelles in charge of producing energy for the cells, and their malfunction has been linked to cardiovascular disease and heart failure. Interestingly, heart mitochondrial content and functionality are also age and sex dependent. Here we investigated the combinatory effects of age and sex in mitochondrial bioenergetics that could help to understand their role on cardiac disease. Cardiac mitochondria from 6- and 24-month-old male and female Wistar rats were isolated, and the enzymatic activities of the oxidative-phosphorylative complexes I, III, and IV and ATPase, as well as the protein levels of complex IV, β-ATPase, and mitochondrial transcription factor A (TFAM), were measured. Furthermore, heart DNA content, citrate synthase activity, mitochondrial protein content, oxygen consumption, and H2O2 generation were also determined. Results showed a reduction in heart mitochondrial mass and functionality with age that correlated with increased H2O2 generation. Moreover, sex-dependent differences were found in several of these parameters. In particular, old females exhibited a significant loss of mitochondrial function and increased relative H2O2 production compared with their male counterparts. The results demonstrate a sex dimorphism in the age-associated defects on cardiac mitochondrial function.

Pinocembrin Protects SH-SY5Y Cells Against MPP+-Induced Neurotoxicity Through the Mitochondrial Apoptotic Pathway

Pinocembrin (PB), the most abundant flavonoid in propolis, has been proven to have neuroprotective property against neurotoxicity in vivo and in vitro. Our recent study demonstrated the neuroprotective effect of PB against Aβ25-35-induced SH-SY5Y neurotoxicity. However, the mechanism as how PB can induce neuroprotection is not known. In the present study, we demonstrate here that PB abrogates the effects of the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) which mimics Parkinson's disease (PD) with elevation of intracellular reactive oxygen species (ROS) level and apoptotic death. We found that pretreatment of SH-SY5Y cells with PB significantly reduced the MPP(+)-induced loss of cell viability, the generation of intracellular ROS, apoptotic rate, and the cleavage of caspase-3. PB strikingly inhibited MPP(+)-induced mitochondrial dysfunctions, including lowered membrane potential, decreased Bcl-2/Bax ratio, and the release of cytochrome c. Overall, these results suggest that PB is intimately involved in inhibiting MPP(+)-induced loss of mitochondrial function and induction of apoptosis that contributes toward neuronal survival. These data indicated that PB might provide a valuable therapeutic strategy for the treatment of PD.

Effects of Regional Mitochondrial Depolarization on Electrical Propagation

Sudden cardiac death often involves arrhythmias triggered by metabolic stress. Loss of mitochondrial function is thought to contribute to the arrhythmogenic substrate, but how mitochondria contribute to uncoordinated electrical activity is poorly understood. It has been proposed that the formation of metabolic current sinks, caused by the nonuniform collapse of mitochondrial inner membrane potential (ΔΨm), contributes to re-entrant arrhythmias because ΔΨm depolarization is tightly coupled to the activation of sarcolemmal ATP-sensitive K(+) channels, hastening action potential repolarization and shortening the refractory period. Here, we use computational and experimental methods to investigate how ΔΨm instability can induce re-entrant arrhythmias. We develop the first tissue-level model of cardiac electrical propagation incorporating cellular electrophysiology, excitation-contraction coupling, mitochondrial energetics, and reactive oxygen species balance. Simulations show that re-entry and fibrillation can be initiated by regional ΔΨm loss because of the disparity of refractory periods inside and outside the metabolic sink. Computational results are compared with the effects of a metabolic sink generated experimentally by local perfusion of a mitochondrial uncoupler in a monolayer of cardiac myocytes. The results demonstrate that regional mitochondrial depolarization triggered by oxidative stress activates sarcolemmal ATP-sensitive K(+) currents to form a metabolic sink. Consequent shortening of the action potential inside, but not outside, the sink increases the propensity for re-entry. ΔΨm recovery during pacing can lead to novel mechanisms of ectopic activation. The findings highlight the importance of mitochondria as potential therapeutic targets for sudden death associated with cardiovascular disease.

A study on aconitase-2 expression change and possible cryo-injury mechanisms after human sperm cryo-thawed

Objective To investigate the change of aconitase-2 (ACO2) between pre-freeze and post-thaw and possible mechanism of cryo-injury.Methods The parameters of sperm from 15 donaters were analyzed by computer-aided sperm analysis (CASA) and revealed a loss in motility; the Western blotting revealed a reduction of ACO2 content after thawing and ACO2 was located in mitochondrial shealth by immunofluorescence test.The change of adenosine-triphosphate (ATP) contents was determined by Chemiluminescence test.The sperm mitochondrial membrane potential was determined by JC-1 test and indirectly revealed mitochondrial function loss by red/green fluorescence,and quantification of fluorescence was determined by Flow Cytometry.T-test was used to compare these two groups.Results Compared with prefreezing group,the motility decreased 18％,ATP contents decreased 32％ and R1 proportion reduced by 27％ (P ＜ 0.05).Conclusion ACO2,degraded after thawing and resulted in mitochandrial functional iniury and reduction in ATP contents,was responsible for sperm motility loss. Key words: Sperm, human; Cryopreservation; Thawing; Aconitase-2 ; Injury

Effect of Australian Propolis from Stingless Bees (Tetragonula carbonaria) on Pre-Contracted Human and Porcine Isolated Arteries

Bee propolis is a mixture of plant resins and bee secretions. While bioactivity of honeybee propolis has been reported previously, information is limited on propolis from Australian stingless bees (Tetragonula carbonaria). The aim of this study was to investigate possible vasomodulatory effects of propolis in KCl-precontracted porcine coronary arteries using an ex vivo tissue bath assay. Polar extracts of propolis produced a dose-dependent relaxant response (EC50=44.7±7.0 μg/ml), which was unaffected by endothelial denudation, suggesting a direct effect on smooth muscle. Propolis markedly attenuated a contractile response to Ca2+ in vessels that were depolarised with 60 mM KCl, in Ca2+-free Krebs solution. Propolis (160 µg/ml) reduced vascular tone in KCl pre-contracted vessels to near-baseline levels over 90 min, and this effect was partially reversible with 6h washout. Some loss in membrane integrity, but no loss in mitochondrial function was detected after 90 min exposure of human cultured umbilical vein endothelial cells to 160 µg/ml propolis. We conclude that Australian stingless bee (T. carbonaria) propolis relaxes porcine coronary artery in an endothelial-independent manner that involves inhibition of voltage-gated Ca2+ channels. This effect is partially and slowly reversible upon washout. Further studies are required to determine the therapeutic potential of Australian stingless bee propolis for conditions in which vascular supply is compromised.

β-Adrenergic-stimulated l-type channel Ca2 + entry mediates hypoxic Ca2 + overload in intact heart

Ca2+ mishandling plays a key role in ischemia- and hypoxia-related cardiac dysfunction and injury. However, the cellular and molecular mechanisms underlying hypoxic intracellular Ca2+ ([Ca2+]i) overload remain incompletely understood. This study aimed to investigate possible mechanisms of [Ca2+]i overload during hypoxia in the intact heart. In Langendorff-perfused heart expressing the Ca2+ indicator GCaMP2, confocal microscopy was used to simultaneously visualize [Ca2+]i, mitochondrial membrane potential (ΔΨm, by tetramethylrhodamine methyl ester) and sarcolemmal integrity (by Evans blue). Upon hypoxia (pO2 ~20mmHg in glucose-free perfusate), [Ca2+]i transients were initially enhanced and then became depressed, arrhythmic, and completely abolished within 12min. At ~20min, basal [Ca2+]i rose to its first peak at a supraphysiological level, coincident with loss of ΔΨm and onset of rigor. A greater [Ca2+]i rise occurred at ~2h and was linked to the loss of sarcolemmal integrity. Removal of extracellular Ca2+ or blockade of the l-type Ca2+ channel (LTCC) (10μM diltiazem or nifedipine) prevented [Ca2+]i overload and markedly delayed the loss of ΔΨm; by contrast, depletion of the sarcoplasmic reticulum Ca2+ store by thapsigargin did not have any significant effect. Importantly, β-adrenergic blockade or depletion of the sympathetic catecholamine store by reserpine slowed the Ca2+ and mitochondrial responses to hypoxia in intact heart. This LTCC-mediated hypoxic [Ca2+]i overload was reproduced in isolated cardiomyocytes when β-adrenergic agonist was present. Taken together, we conclude that Ca2+ entry through β-adrenergic-stimulated LTCC underlies hypoxia-induced [Ca2+]i overload and the ensuing loss of mitochondrial function in intact heart.