Emerging findings suggest that two lineages of mitochondrial Ca(2+) uptake participate during active and resting states: 1) the major eukaryotic membrane potential-dependent mitochondrial Ca(2+) ...uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca(2+) across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca(2+) accumulation are unclear. Solute carriers--solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25--represent a family of EF-hand-containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference-mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca(2+) uptake and reduces cytosolic Ca(2+) clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand-domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca(2+) uptake. In addition, SLC25A23 interacts with mitochondrial Ca(2+) uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing IMCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA-insensitive SLC25A23 cDNA restores mitochondrial Ca(2+) uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca(2+) influx.
In adult left ventricular mouse myocytes, exposure to sodium cyanide (NaCN) in the presence of glucose dose-dependently reduced contraction amplitude, with ~80% of maximal inhibitory effect attained ...at 100 µM. NaCN (100 µM) exposure for 10 min significantly decreased contraction and intracellular Ca
concentration (Ca
) transient amplitudes, systolic but not diastolic Ca
, and maximal L-type Ca
current ( I
) amplitude, indicating acute alteration of Ca
homeostasis largely accounted for the observed excitation-contraction abnormalities. In addition, NaCN depolarized resting membrane potential ( E
), reduced action potential (AP) amplitude, prolonged AP duration at 50% (APD
) and 90% repolarization (APD
), and suppressed depolarization-activated K
currents but had no effect on Na
-Ca
exchange current ( I
). NaCN did not affect cellular adenosine triphosphate levels but depolarized mitochondrial membrane potential (ΔΨ
) and increased superoxide (O
) levels. Methylene blue (MB; 20 µg/ml) added 3 min after NaCN restored contraction and Ca
transient amplitudes, systolic Ca
, E
, AP amplitude, APD
, APD
, I
, depolarization-activated K
currents, ΔΨ
, and O
levels toward normal. We conclude that MB reversed NaCN-induced cardiotoxicity by preserving intracellular Ca
homeostasis and excitation-contraction coupling ( I
), minimizing risks of arrhythmias ( E
, AP configuration, and depolarization-activated K
currents), and reducing O
levels. NEW & NOTEWORTHY Cyanide poisoning due to industrial exposure, smoke inhalation, and bioterrorism manifests as cardiogenic shock and requires rapidly effective antidote. In the early stage of cyanide exposure, adenosine triphosphate levels are normal but myocyte contractility is reduced, largely due to alterations in Ca
homeostasis because of changes in oxidation-reduction environment of ion channels. Methylene blue, a drug approved by the U.S. Food and Drug Administration, ameliorates cyanide toxicity by normalizing oxidation-reduction state and Ca
channel function.
G protein-coupled receptor kinases (GRKs) phosphorylate activated receptors to promote arrestin binding, decoupling from heterotrimeric G proteins, and internalization. GRK2 and GRK5 are ...overexpressed in the failing heart and thus have become therapeutic targets. Previously, we discovered two classes of GRK2-selective inhibitors, one stemming from GSK180736A, a Rho-associated coiled-coil containing kinase 1 (ROCK1) inhibitor, the other from paroxetine, a selective serotonin-reuptake inhibitor. These two classes of compounds bind to the GRK2 active site in a similar configuration but contain different hinge-binding "warheads": indazole and benzodioxole, respectively. We surmised from our prior studies that an indazole would be the stronger hinge binder and would impart increased potency when substituted for benzodioxole in paroxetine derivatives. To test this hypothesis, we synthesized a series of hybrid compounds that allowed us to compare the effects of inhibitors that differ only in the identity of the warhead. The indazole-paroxetine analogs were indeed more potent than their respective benzodioxole derivatives but lost selectivity. To investigate how these two warheads dictate selectivity, we determined the crystal structures of three of the indazole hybrid compounds (CCG224061, CCG257284, and CCG258748) in complex with GRK2-G
Comparison of these structures with those of analogous benzodioxole-containing complexes confirmed that the indazole-paroxetine hybrids form stronger interactions with the hinge of the kinase but also stabilize a distinct conformation of the kinase domain of GRK2 compared with previous complexes with paroxetine analogs. This conformation is analogous to one that can be assumed by GRK5, at least partially explaining the loss in selectivity.
The pathophysiology of human immunodeficiency virus (HIV)‐associated cardiomyopathy remains uncertain. We used HIV‐1 transgenic (Tg26) mice to explore mechanisms by which HIV‐related proteins ...impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type WT) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨ
m) under normoxic conditions but lower Δ
Ψ
m after hypoxia/reoxygenation (H/R). In addition, Δ
Ψ
m in Tg26 myocytes failed to recover after Ca
2+ challenge. Functionally, mitochondrial Ca
2+ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post‐H/R, mitochondrial superoxide (O
2
•–) levels were higher in Tg26 compared to WT myocytes. Overexpression of B‐cell lymphoma 2‐associated athanogene 3 (BAG3) reduced O
2
•– levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post‐H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post‐H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein
Tat. We conclude: (a) Under basal conditions, mitochondrial Ca
2+ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post‐H/R, Δ
Ψ
m was lower, mitochondrial O
2
•– levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O
2
•– levels and restored contraction amplitudes to normal in Tg26 myocytes post‐H/R in the presence of isoproterenol.
The hemizygous NL4‐3 Δgag/pol transgenic (Tg26) mouse with replication‐deficient, noninfectious human immunodeficiency virus type 1 (HIV‐1) but with HIV‐1 proteins present in tissues simulates contemporary HIV‐1‐infected patients treated with combination antiretroviral therapy and is an excellent model to study chronic complications of HIV‐1 infection such as HIV‐associated cardiomyopathy. Cardiac myocytes isolated from Tg26 mice demonstrate mitochondrial dysfunction and elevated reactive oxygen species (ROS) that are exacerbated by hypoxia/reoxygenation and adrenergic stress. B‐cell lymphoma 2‐associated athanogene 3 (BAG3) regulates autophagy, apoptosis, excitation‐contraction coupling, mitochondrial quality control, and sarcomere stability in cardiac myocytes and can associate with the HIV‐1 protein Tat. Overexpression of BAG3 in Tg26 myocytes restores the elevated ROS levels to normal and improves contractile dysfunction. BAG3 may be a useful therapeutic modality in the treatment of HIV‐associated cardiomyopathy.
Ubiquitously expressed Trpm2 channel limits oxidative stress and preserves mitochondrial function. We first demonstrated that intracellular Ca(2+) concentration increase after Trpm2 activation was ...due to direct Ca(2+) influx and not indirectly via reverse Na(+)/Ca(2+) exchange. To elucidate whether Ca(2+) entry via Trpm2 is required to maintain cellular bioenergetics, we injected adenovirus expressing green fluorescent protein (GFP), wild-type (WT) Trpm2, and loss-of-function (E960D) Trpm2 mutant into left ventricles of global Trpm2 knockout (gKO) or WT hearts. Five days post-injection, gKO-GFP heart slices had higher reactive oxygen species (ROS) levels but lower oxygen consumption rate (OCR) than WT-GFP heart slices. Trpm2 but not E960D decreased ROS and restored OCR in gKO hearts back to normal levels. In gKO myocytes expressing Trpm2 or its mutants, Trpm2 but not E960D reduced the elevated mitochondrial superoxide (O2(.-)) levels in gKO myocytes. After hypoxia-reoxygenation (H/R), Trpm2 but not E906D or P1018L (inactivates Trpm2 current) lowered O2(.-) levels in gKO myocytes and only in the presence of extracellular Ca(2+), indicating sustained Ca(2+) entry is necessary for Trpm2-mediated preservation of mitochondrial function. After ischemic-reperfusion (I/R), cardiac-specific Trpm2 KO hearts exhibited lower maximal first time derivative of LV pressure rise (+dP/dt) than WT hearts in vivo. After doxorubicin treatment, Trpm2 KO mice had worse survival and lower +dP/dt. We conclude 1) cardiac Trpm2-mediated Ca(2+) influx is necessary to maintain mitochondrial function and protect against H/R injury; 2) Ca(2+) influx via cardiac Trpm2 confers protection against H/R and I/R injury by reducing mitochondrial oxidants; and 3) Trpm2 confers protection in doxorubicin cardiomyopathy.
Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. ...Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.
Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including ...neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.
Background The use of a guidewire (GW) for cannulation of the bile duct during ERCP may prevent post-ERCP pancreatitis (PEP). Objectives A systematic review and meta-analysis of GW-guided versus ...conventional contrast (CC)–guided bile duct cannulation for the prevention PEP. Design A November 2008 search of gray literature, databases, reference lists, and meeting abstracts was conducted for randomized, controlled trials comparing GW and CC. Two independent reviewers extracted the data. The outcomes included PEP, primary cannulation success, and other adverse events. Results From 2132 citations, 7 randomized, controlled trials (5 noncrossover trials and 2 crossover trials) were included. Among noncrossover trials only, there was significant reduction in PEP when using a GW (3.2%) compared with CC (8.7%) (relative risk RR 0.38; 95% CI, 0.19-0.76). Subgroup analysis showed a significantly lower occurrence of PEP after GW entry versus CC injection of the pancreatic duct (1.1% vs 9.5%; RR 0.19; 95% CI, 0.06-0.58). Among patients with a precut sphincterotomy from a failed primary cannulation, there was less PEP with GW cannulation compared with CC (2.4% vs 21.7%; RR 0.21; 95% CI, 0.04-1.04). The other adverse event rates were comparable between GW and CC groups (2% vs 2%; RR 1.05; 95% CI, 0.39-2.83). Primary cannulation success was significantly greater with GW use compared with CC (89% vs 78%; RR 1.19; 95% CI, 1.05-1.35). Conclusion ERCP GW cannulation reduces the risk of PEP compared with the use of CC. GW cannulation is associated with a higher cannulation success rate and less PEP after pancreatic duct entry.
BACKGROUND—The incidence and impact of clinical stroke and silent radiographic cerebral infarction complicating open surgical aortic valve replacement (AVR) are poorly characterized.
METHODS AND ...RESULTS—We performed a prospective cohort study of subjects ≥65 years of age who were undergoing AVR for calcific aortic stenosis. Subjects were evaluated by neurologists preoperatively and postoperatively and underwent postoperative magnetic resonance imaging. Over a 4-year period, 196 subjects were enrolled at 2 sites (mean age, 75.8±6.2 years; 36% women; 6% nonwhite). Clinical strokes were detected in 17%, transient ischemic attack in 2%, and in-hospital mortality was 5%. The frequency of stroke in the Society for Thoracic Surgery database in this cohort was 7%. Most strokes were mild; the median National Institutes of Health Stroke Scale was 3 (interquartile range, 1–9). Clinical stroke was associated with increased length of stay (median, 12 versus 10 days; P=0.02). Moderate or severe stroke (National Institutes of Health Stroke Scale ≥10) occurred in 8 (4%) and was strongly associated with in-hospital mortality (38% versus 4%; P=0.005). Of the 109 stroke-free subjects with postoperative magnetic resonance imaging, silent infarct was identified in 59 (54%). Silent infarct was not associated with in-hospital mortality or increased length of stay.
CONCLUSIONS—Clinical stroke after AVR was more common than reported previously, more than double for this same cohort in the Society for Thoracic Surgery database, and silent cerebral infarctions were detected in more than half of the patients undergoing AVR. Clinical stroke complicating AVR is associated with increased length of stay and mortality.
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