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1.  Mitochondria-Derived Superoxide Links to Tourniquet-Induced Apoptosis in Mouse Skeletal Muscle 
PLoS ONE  2012;7(8):e43410.
Our previous study has reported that superoxide mediates ischemia-reperfusion (IR)-induced necrosis in mouse skeletal muscle. However, it remains poorly understood whether IR induces apoptosis and what factors are involved in IR-induced apoptosis in skeletal muscle. Using a murine model of tourniquet-induced hindlimb IR, we investigated the relationship between mitochondrial dysfunction and apoptosis in skeletal muscle. Hindlimbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Compared to sham treatment, tourniquet-induced IR significantly elevated mitochondria-derived superoxide production, activated opening of mitochondrial permeability transition pore (mPTP), and caused apoptosis in the gastrocnemius muscles. Pretreatment with a superoxide dismutase mimetic (tempol, 50 mg/kg) or a mitochondrial antioxidant (co-enzyme Q10, 50 mg/kg) not only decreased mitochondria-derived superoxide production, but also inhibited mPTP opening and apoptosis in the IR gastrocnemius muscles. Additionally, an inhibitor of mPTP (cyclosporine A, 50 mg/kg) also inhibited both mPTP opening and apoptosis in the IR gastrocnemius muscles. These results suggest that mitochondria-derived superoxide overproduction triggers the mPTP opening and subsequently causes apoptosis in tourniquet-induced hindlimb IR.
PMCID: PMC3422247  PMID: 22912870
2.  Reduced hind limb ischemia-reperfusion injury in Toll-like receptor-4 mutant mice is associated with decreased neutrophil extracellular traps 
Journal of vascular surgery  2013;58(6):10.1016/j.jvs.2013.02.241.
Ischemia-reperfusion (IR) injury is a significant problem in the management of patients with acute limb ischemia (ALI). Despite rapid restoration of blood flow following technically successful open and endovascular revascularization, complications secondary to IR injury continue to occur and limit clinical success. Our aim was to create a murine model of hind limb IR injury to examine the role of Toll-like receptor-4 (TLR4) and to determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which is known to be highly thrombogenic and may mediate microvascular injury.
A calibrated tension tourniquet was applied to unilateral hind limb of wild type (WT) and TLR4 receptor mutant (TLR4m) mice for 1.5 hours to induce ischemia and then immediately removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were sacrificed and hind limb tissue as well as serum specimens were collected for analysis. Hematoxylin and eosin stained sections of hind limb skeletal muscle tissue were examined for fiber injury. For immunohistochemistry, mouse monoclonal anti-histone H2A/H2B/DNA complex antibody to detect NETs and rabbit polyclonal anti-myeloperoxidase (MPO) antibody were used to identify infiltrating cells containing MPO. Muscle ATP levels, nuclear NF-κB activity, IκBα, poly (ADP-ribose) polymerase (PARP) activity and iNOS expression were measured. Systemic levels of KC, MCP-1 and VEGF in the serum samples were also examined.
IR injury in the hind limb of wild type mice demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation, decreased I-κBα levels, increased iNOS expression and increased PARP activity levels when compared to the TLR4 knockout mice samples. Additionally, there was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4 mutant mice, which corresponded to similar levels of decreased NETs detection in the interstitial space and in microvascular thrombi. In situ nuclease treatment of wild-type tissue sections significantly diminished the level of NETs immunostaining demonstrating the specificity of our antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury.
These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may contribute to muscle fiber injury.
PMCID: PMC3825746  PMID: 23683381
3.  Remote preconditioning, perconditioning, and postconditioning: a comparative study of their cardioprotective properties in rat models 
Clinics  2013;68(2):263-268.
Ischemia reperfusion injury is partly responsible for the high mortality associated with induced myocardial injury and the reduction in the full benefit of myocardial reperfusion. Remote ischemic preconditioning, perconditioning, and postconditioning have all been shown to be cardioprotective. However, it is still unknown which one is the most beneficial. To examine this issue, we used adult male Wistar rat ischemia reperfusion models to compare the cardioprotective effect of these three approaches applied on double-sided hind limbs.
The rats were randomly distributed to the following five groups: sham, ischemia reperfusion, remote preconditioning, remote perconditioning, and remote post-conditioning. The ischemia/reperfusion model was established by sternotomy followed by a 30-min ligation of the left coronary artery and a subsequent 3-h reperfusion. Remote conditioning was induced with three 5-min ischemia/5-min reperfusion cycles of the double-sided hind limbs using a tourniquet.
A lower early reperfusion arrhythmia score (1.50±0.97) was found in the rats treated with remote perconditioning compared to those in the ischemia reperfusion group (2.33±0.71). Meanwhile, reduced infarct size was also observed (15.27±5.19% in remote perconditioning, 14.53±3.45% in remote preconditioning, and 19.84±5.85% in remote post-conditioning vs. 34.47±7.13% in ischemia reperfusion, p<0.05), as well as higher expression levels of the apoptosis-relevant protein Bcl-2/Bax following global (ischemia/reperfusion) injury in in vivo rat heart models (1.255±0.053 in remote perconditioning, 1.463±0.290 in remote preconditioning, and 1.461±0.541 in remote post-conditioning vs. 1.003±0.159 in ischemia reperfusion, p<0.05).
Three remote conditioning strategies implemented with episodes of double-sided hind limb ischemia/reperfusion have similar therapeutic potential for cardiac ischemia/reperfusion injury, and remote perconditioning has a greater ability to prevent reperfusion arrhythmia.
PMCID: PMC3584272  PMID: 23525325
Cardioprotective Property; Ischemia/Reperfusion Injury; Models
4.  The effect of Antithrombin-III on routine hematological and biochemical parameters in an experimental animal model of skeletal muscle ischemia-reperfusion injury 
Hippokratia  2014;18(3):234-239.
Antithrombin III (AT-III) has been shown to attenuate the local and systemic harmful effects of skeletal muscle ischemia-reperfusion (I-R) injury. The aim of the present study was to monitor the fluctuation of routine hematological and biochemical parameters in an experimental animal model of tourniquet-induced skeletal muscle I-R injury and to investigate how these are influenced by the protective administration of AT-III.
Sixty male Wistar rats were submitted to a 6-hour, tourniquet-induced, complete ischemia of the right hind-limb. Animals were divided into those receiving AT-III (dose, 250 IU/kg) 30 minutes before the reperfusion (group A, n=30) and those receiving placebo (group B, n =30). Another 10 animals were sham-operated (group C). White blood cell (WBC) and platelet (PLT) count, aspartate and alanine aminotransferases (AST and ALT), alkaline phosphatase (ALP), and γ-glutamyl transferase (γ-GT) were estimated in blood samples taken from the inferior vena cava at 3 different time points post-reperfusion (at baseline, at 30 minutes and at 4 hours) and groups A and B were compared using the Mann-Whitney U test.
There were no statistically significant differences between the AT-III and the placebo groups at 0, 30 minutes and 4 hours with regard to the WBC, ALT and γ-GT levels, however, there was a significant decrease of AST levels 4 hours post-reperfusion in the AT-III group compared to the placebo group (p=0.002). An increased PLT count and ALP levels 30 minutes post-reperfusion were also noted in the AT-III group compared to placebo (p<0.001; and p=0.001, respectively).
Of the routine hematological and biochemical parameters tested, AST was found to be significantly suppressed at 4 hours in the AT-III-treated animals, suggesting a possible beneficial effect of AT-III in mouse skeletal muscle I-R injury. The effect of AT-III on PLTs and ALP levels merits further investigation. Hippokratia 2014; 18 (3): 234-239.
PMCID: PMC4309144
Experimental study; lower limb; ischemia-reperfusion; skeletal muscle; antithrombin-III
5.  Hydrogen sulfide reduces neutrophil recruitment in hind-limb ischemia-reperfusion injury in an L-selectin and ADAM-17 dependent manner 
Plastic and reconstructive surgery  2013;131(3):487-497.
Reperfusion following ischemia leads to neutrophil recruitment injured tissue. Selectins and β2 integrins regulate neutrophil interaction with the endothelium during neutrophil rolling and firm adhesion. Excessive neutrophil infiltration into tissue is thought to contribute to IRI damage. NaHS mitigates the damage caused by ischemia-reperfusion injury (IRI). This study's objective was to determine the effect of hydrogen sulfide (NaHS) on neutrophil adhesion receptor expression.
Human neutrophils were either left untreated or incubated in 20 μM NaHS, and/or 50 μg/mL pharmacological ADAM-17 inhibitor TAPI-0; activated by IL-8, fMLP, or TNF-α; and labeled against PSGL-1, LFA-1, Mac-1 α, L-selectin and β2 integrin epitopes CBRM1/5 or KIM127 for flow cytometry. Cohorts of 3 C57BL/6 mice received an intravenous dose of saline vehicle, or 20 μM NaHS with or without 50 μg/mL TAPI-0 before unilateral tourniquet induced hind-limb ischemia for 3 hours followed by 3 hours of reperfusion. Bilateral gastrocnemius muscles were processed for histology before neutrophil infiltration quantification.
NaHS treatment significantly increased L-selectin shedding from human neutrophils following activation by fMLP and IL-8 in an ADAM-17 dependent manner. Mice treated with NaHS to raise bloodstream concentration by 20 μM prior to ischemia or reperfusion showed a significant reduction in neutrophil recruitment into skeletal muscle tissue following tourniquet-induced hindlimb IRI.
NaHS administration results in the downregulation of L-selectin expression in activated human neutrophils. This leads to a reduction in neutrophil extravasation and tissue infiltration and may partially account for the protective effects of NaHS seen in the setting of IRI.
PMCID: PMC3587315  PMID: 23446563
6.  Restoration of skeletal muscle ischemia-reperfusion injury in humanized immunodeficient mice 
Surgery  2009;146(2):340-346.
Ischemia and reperfusion (I/R) of tissue provokes an inflammatory process that is highly dependent on circulating natural immunoglobulin M (IgM) and the complement cascade. In mice, a single IgM specificity produced by peritoneal B cells can initiate reperfusion injury. It is unknown whether humans express natural IgM with a similar specificity. It is also unknown whether pathogenic IgM is produced solely from peritoneal B cells or can also be made by circulating B cells.
Immunodeficient mice lacking endogenous immunoglobulin were used. Mice were reconstituted with normal saline, human serum, or xenografted human peripheral blood lymphocytes (PBLs) and then subjected to tourniquet induced hindlimb ischemia and reperfusion. Serum human IgM and IgG were measured by ELISA. Skeletal muscle was harvested for injury assessment by histology and for immunohistochemistry.
Immunodeficient mice are protected from skeletal muscle injury following hindlimb I/R. Transfer of human serum restores skeletal muscle damage. Rag2/γR -/- mice engrafted with human PBL (huPBL-SCID) have high levels of human IgM. huPBL-SCID mice develop significantly more skeletal muscle injury than control saline treated (p ≤ 0.01) and human serum reconstituted Rag2/γR -/- mice (p ≤ 0.01). Sham treated huPBL-SCID mice have no muscle injury, demonstrating that human lymphocyte engraftment does not cause injury in the absence of ischemia. Deposition of human IgM is seen on injured but not sham injured muscle.
Human serum can initiate murine skeletal muscle ischemia reperfusion injury. Circulating human PBL may be a source of pathogenic IgM. The huPBL-SCID mouse may be a useful model to define the specificity of pathogenic human IgM and to test therapeutics for ischemia-reperfusion injury.
PMCID: PMC2767199  PMID: 19628094
7.  Extracellular BCL2 Proteins Are Danger-Associated Molecular Patterns That Reduce Tissue Damage in Murine Models of Ischemia-Reperfusion Injury 
PLoS ONE  2010;5(2):e9103.
Ischemia-reperfusion (I/R) injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs) released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system.
Methodology/Principal Findings
Ischemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rh)BCL2 protein or rhBCL2-related protein A1 (BCL2A1) (50 ng/g) given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2) protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2) or the adaptor protein, myeloid differentiation factor-88 (MyD88).
Treatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.
PMCID: PMC2816997  PMID: 20161703
8.  Effects of penehyclidine hydrochloride in small intestinal damage caused by limb ischemia-reperfusion 
AIM: To investigate the protective effect of penehyclidine hydrochloride post-conditioning in the damage to the barrier function of the small intestinal mucosa caused by limb ischemia-reperfusion (LIR) injury.
METHODS: Male Wistar rats were randomly divided into three groups (36 rats each): the sham-operation group (group S), lower limb ischemia-reperfusion group (group LIR), and penehyclidine hydrochloride post-conditioning group (group PHC). Each group was divided into subgroups (n = 6 in each group) according to ischemic-reperfusion time, i.e. immediately 0 h (T1), 1 h (T2), 3 h (T3), 6 h (T4), 12 h (T5), and 24 h (T6). Bilateral hind-limb ischemia was induced by rubber band application proximal to the level of the greater trochanter for 3 h. In group PHC, 0.15 mg/kg of penehyclidine hydrochloride was injected into the tail vein immediately after 3 h of bilateral hind-limb ischemia. The designated rats were sacrificed at different time-points of reperfusion; diamine oxidase (DAO), superoxide dismutase (SOD) activity, myeloperoxidase (MPO) of small intestinal tissue, plasma endotoxin, DAO, tumor necrosis factor-α (TNF-α), and interleukin (IL)-10 in serum were detected in the rats.
RESULTS: The pathological changes in the small intestine were observed under light microscope. The levels of MPO, endotoxin, serum DAO, and IL-10 at T1-T6, and TNF-α level at T1-T4 increased in groups LIR and PHC (P < 0.05) compared with those in group S, but tissue DAO and SOD activity at T1-T6 decreased (P < 0.05). In group PHC, the tissue DAO and SOD activity at T2-T6, and IL-10 at T2-T5 increased to higher levels than those in group LIR (P < 0.05); however, the levels of MPO, endotoxin, and DAO in the blood at T2-T6, and TNF-α at T2 and T4 decreased (P < 0.05).
CONCLUSION: Penehyclidine hydrochloride post-conditioning may reduce the permeability of the small intestines after LIR. Its protection mechanisms may be related to inhibiting oxygen free radicals and inflammatory cytokines for organ damage.
PMCID: PMC3020382  PMID: 21246001
Penehyelidine hydrochloride; Post-conditioning; Limb ischemia-reperfusion injury; Small intestine; Protection
9.  Putative role of ischemic postconditioning in a rat model of limb ischemia and reperfusion: involvement of hypoxia-inducible factor-1α expression 
Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It improves angiogenesis and tissue perfusion in ischemic skeletal muscle. In the present study, we tested the hypothesis that ischemic postconditioning is effective for salvaging ischemic skeletal muscle resulting from limb ischemia-reperfusion injury, and that the mechanism involves expression of HIF-1α. Wistar rats were randomly divided into three groups (n=36 each): sham-operated (group S), hindlimb ischemia-reperfusion (group IR), and ischemic postconditioning (group IPO). Each group was divided into subgroups (n=6) according to reperfusion time: immediate (0 h, T0), 1 h (T1), 3 h (T3), 6 h (T6), 12 h (T12), and 24 h (T24). In the IPO group, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were carried out before reperfusion. At all reperfusion times (T0-T24), serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, as well as interleukin (IL)-6, IL-10, and tumor necrosis factor-α (TNF-α) concentrations, were measured in rats after they were killed. Histological and immunohistochemical methods were used to assess the skeletal muscle damage and HIF-1α expression in skeletal muscle ischemia. In groups IR and IPO, serum LDH and CK activities and TNF-α, IL-6, and IL-10 concentrations were all significantly increased compared to group S, and HIF-1α expression was up-regulated (P<0.05 or P<0.01). In group IPO, serum LDH and CK activities and TNF-α and IL-6 concentrations were significantly decreased, IL-10 concentration was increased, HlF-1α expression was down-regulated (P<0.05 or P<0.01), and the pathological changes were reduced compared to group IR. The present study suggests that ischemic postconditioning can reduce skeletal muscle damage caused by limb ischemia-reperfusion and that its mechanisms may be related to the involvement of HlF-1α in the limb ischemia-reperfusion injury-triggered inflammatory response.
PMCID: PMC4143200  PMID: 25075575
Hypoxia-inducible factor-1α; Ischemic postconditioning; Skeletal muscle ischemia
10.  The role of mast cells and fibre type in ischaemia reperfusion injury of murine skeletal muscles 
Ischaemia reperfusion (IR) injury of skeletal muscle, is a significant cause of morbidity following trauma and surgical procedures, in which muscle fibre types exhibit different susceptibilities. The relative degree of mast cell mediated injury, within different muscle types, is not known.
In this study we compared susceptibility of the fast-twitch, extensor digitorum longus (EDL), mixed fast/slow-twitch gastrocnemius and the predominately slow-twitch soleus, muscles to ischemia reperfusion (IR) injury in four groups of mice that harbour different mast cell densities; C57/DBA mast cell depleted (Wf/Wf), their heterozygous (Wf/+) and normal littermates (+/+) and control C57BL/6 mice. We determined whether susceptibility to IR injury is associated with mast cell content and/or fibre type and/or mouse strain. In experimental groups, the hind limbs of mice were subjected to 70 minutes warm tourniquet ischemia, followed by 24 h reperfusion, and the muscle viability was assessed on fresh whole-mount slices by the nitroblue tetrazolium (NBT) histochemical assay.
Viability was remarkably higher in the Wf/Wf strain irrespective of muscle type. With respect to muscle type, the predominately slow-twitch soleus muscle was significantly more resistant to IR injury than gastrocnemius and the EDL muscles in all groups. Mast cell density was inversely correlated to muscle viability in all types of muscle.
These results show that in skeletal muscle, IR injury is dependent upon both the presence of mast cells and on fibre type and suggest that a combination of preventative therapies may need to be implemented to optimally protect muscles from IR injury.
PMCID: PMC1074344  PMID: 15813978
11.  Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/reperfusion induced apoptosis and autophagy in the rat 
Prolonged ischemia amplified iscehemia/reperfusion (IR) induced renal apoptosis and autophagy. We hypothesize that ischemic conditioning (IC) by a briefly intermittent reperfusion during a prolonged ischemic phase may ameliorate IR induced renal dysfunction. We evaluated the antioxidant/oxidant mechanism, autophagy and apoptosis in the uninephrectomized Wistar rats subjected to sham control, 4 stages of 15-min IC (I15 × 4), 2 stages of 30-min IC (I30 × 2), and total 60-min ischema (I60) in the kidney followed by 4 or 24 hours of reperfusion. By use of ATP assay, monitoring O2-. amounts, autophagy and apoptosis analysis of rat kidneys, I60 followed by 4 hours of reperfusion decreased renal ATP and enhanced reactive oxygen species (ROS) level and proapoptotic and autophagic mechanisms, including enhanced Bax/Bcl-2 ratio, cytochrome C release, active caspase 3, poly-(ADP-ribose)-polymerase (PARP) degradation fragments, microtubule-associated protein light chain 3 (LC3) and Beclin-1 expression and subsequently tubular apoptosis and autophagy associated with elevated blood urea nitrogen and creatinine level. I30 × 2, not I15 × 4 decreased ROS production and cytochrome C release, increased Manganese superoxide dismutase (MnSOD), Copper-Zn superoxide dismutase (CuZnSOD) and catalase expression and provided a more efficient protection than I60 against IR induced tubular apoptosis and autophagy and blood urea nitrogen and creatinine level. We conclude that 60-min renal ischemia enhanced renal tubular oxidative stress, proapoptosis and autophagy in the rat kidneys. Two stages of 30-min ischemia with 3-min reperfusion significantly preserved renal ATP content, increased antioxidant defense mechanisms and decreased ischemia/reperfusion enhanced renal tubular oxidative stress, cytosolic cytochrome C release, proapoptosis and autophagy in rat kidneys.
PMCID: PMC2653526  PMID: 19272187
12.  Effects of Ascorbic Acid, Alpha-Tocopherol and Allopurinol on Ischemia-Reperfusion Injury in Rabbit Skeletal Muscle: An Experimental Study 
Drug Target Insights  2007;2:249-258.
Ischemia reperfusion injury to skeletal muscle, following an acute arterial occlusion is important cause of morbidity and mortality. The aim of the present study was to determine and evaluate the effects of ascorbic acide, alpha-tocopherol and allopurinol on ischemia reperfusion injury in rabbit skeletal muscle.
Forty-eight New Zealand white rabbits, all male, weighing between 2.5 to 3.0 (mean 2.8) kg, were used in the study. They were separated into four groups. Group I was the control group without any drugs. The other groups were treatment groups (groups II, III, and IV). Group II rabbits administrated 50 mg/kg ascorbic acide and 100 mg/kg alpha-tocopherol 3 days prior to ischemia, group III rabbits received 50 mg/kg allopurinol 2 days prior to ischemia, and group IV rabbits were administrated both 50 mg/kg ascorbic acide, 100 mg/kg alpha-tocopherol 3 days prior to ischemia and 50 mg/kg allopurinol 2 days prior to ischemia. Two hours ischemia and 2 hours reperfusion were underwent to the treatment groups. At the end of the reperfusion periods, muscle samples were taken from rectus femoris muscle for determination of superoxide dismutase, catalase and glutathione peroxidase activities as antioxidant enzymes, and malondialdehyde as an indicator of lipid peroxidation and xanthine oxidase levels as source hydroxyl radical. Besides, histopathological changes (edema, inflammation, ring formation and splitting formation) were evaluated in the muscle specimens.
In the treatment groups; superoxide dismutase (U/mgprotein), catalase (U/mgprotein), and glutathione peroxidase (U/mgprotein) levels increased, malondialdehyde (nmol/mgprotein) and xanthine oksidase (mU/mgprotein) levels decreased compared to control I ( p < 0.05). Increase of superoxide dismutase, catalase, and glutathione peroxidase levels were the highest and decrease of malondialdehyde and xanthine oxidase levels were the highest in group IV compared to groups II and III, but no significant as statistically. Also amount of cellular injury in group II, III, and IV were lower than group I.
Antioxidant medication may help lowering ischemia reperfusion injury. In our study, all drug medications are shown to be able to have an effective role for preventing ischemia reperfusion injury. Moreover, ascorbic acide + alpha-tocopherol + allopurinol group (group IV) may have a beneficial effect to decrease the local and systemic damage due to ischemia-reperfusion injury.
PMCID: PMC3155224  PMID: 21901079
Ischemia-reperfusion injury; antioxidant agents; ascorbic acid; alpha-tocopherol; allopurinol
13.  C1 Esterase Inhibitor Reduces Lower Extremity Ischemia/Reperfusion Injury and Associated Lung Damage 
PLoS ONE  2013;8(8):e72059.
Ischemia/reperfusion injury of lower extremities and associated lung damage may result from thrombotic occlusion, embolism, trauma, or surgical intervention with prolonged ischemia and subsequent restoration of blood flow. This clinical entity is characterized by high morbidity and mortality. Deprivation of blood supply leads to molecular and structural changes in the affected tissue. Upon reperfusion inflammatory cascades are activated causing tissue injury. We therefore tested preoperative treatment for prevention of reperfusion injury by using C1 esterase inhibitor (C1 INH).
Methods and Findings
Wistar rats systemically pretreated with C1 INH (n = 6), APT070 (a membrane-targeted myristoylated peptidyl construct derived from human complement receptor 1, n = 4), vehicle (n = 7), or NaCl (n = 8) were subjected to 3h hind limb ischemia and 24h reperfusion. The femoral artery was clamped and a tourniquet placed under maintenance of a venous return. C1 INH treated rats showed significantly less edema in muscle (P<0.001) and lung and improved muscle viability (P<0.001) compared to controls and APT070. C1 INH prevented up-regulation of bradykinin receptor b1 (P<0.05) and VE-cadherin (P<0.01), reduced apoptosis (P<0.001) and fibrin deposition (P<0.01) and decreased plasma levels of pro-inflammatory cytokines, whereas deposition of complement components was not significantly reduced in the reperfused muscle.
C1 INH reduced edema formation locally in reperfused muscle as well as in lung, and improved muscle viability. C1 INH did not primarily act via inhibition of the complement system, but via the kinin and coagulation cascade. APT070 did not show beneficial effects in this model, despite potent inhibition of complement activation. Taken together, C1 INH might be a promising therapy to reduce peripheral ischemia/reperfusion injury and distant lung damage in complex and prolonged surgical interventions requiring tourniquet application.
PMCID: PMC3753343  PMID: 23991040
14.  Overexpression of stanniocalcin-1 inhibits reactive oxygen species and renal ischemia/reperfusion injury in mice 
Kidney international  2012;82(8):867-877.
Reactive oxygen species, endothelial dysfunction, inflammation, and mitogen-activated protein kinases have important roles in the pathogenesis of ischemia/reperfusion kidney injury. Stanniocalcin-1 (STC1) suppresses superoxide generation in many systems through induction of mitochondrial uncoupling proteins and blocks the cytokine-induced rise in endothelial permeability. Here we tested whether transgenic overexpression of STC1 protects from bilateral ischemia/reperfusion kidney injury. This injury in wild type mice caused a halving of the creatinine clearance; severe tubular vacuolization and cast formation; increased infiltration of macrophages and T cells; higher vascular permeability; greater production of superoxide and hydrogen peroxide; and higher ratio of activated ERK/activated JNK and p38, all compared to sham-treated controls. Mice transgenic for human STC1 expression, however, had resistance to equivalent ischemia/reperfusion injury indicated as no significant change from controls in any of these parameters. Tubular epithelial cells in transgenic mice expressed higher mitochondrial uncoupling protein 2 and lower superoxide generation. Pre-treatment of transgenic mice with paraquat, a generator of reactive oxygen species, before injury restored the susceptibility to ischemia/reperfusion kidney injury, suggesting that STC1 protects by an anti-oxidant mechanism. Thus, STC1 may be a therapeutic target for ischemia/reperfusion kidney injury.
PMCID: PMC3443530  PMID: 22695329
vascular permeability; mitochondria; free radicals; inflammation
15.  Cardiomyocyte-restricted overexpression of extracellular superoxide dismutase increases nitric oxide bioavailability and reduces infarct size after ischemia/reperfusion 
Basic Research in Cardiology  2012;107(6):305.
Increased levels of extracellular superoxide dismutase (ecSOD) induced by preconditioning or gene therapy protect the heart from ischemia/reperfusion injury. To elucidate the mechanism responsible for this action, we studied the effects of increased superoxide scavenging on nitric oxide (NO) bioavailability in a cardiac myocyte-specific ecSOD transgenic (Tg) mouse. Results indicated that ecSOD overexpression increased cardiac myocyte-specific ecSOD activity 27.5-fold. Transgenic ecSOD was localized to the sarcolemma and, notably, the cytoplasm of cardiac myocytes. Ischemia/reperfusion injury was attenuated in ecSOD Tg hearts, in which infarct size was decreased and LV functional recovery was improved. Using the ROS spin trap, DMPO, electron paramagnetic resonance (EPR) spectroscopy demonstrated a significant decrease in ROS in Tg hearts during the first 20 min of reperfusion. This decrease in ROS was accompanied by an increase in NO production determined by EPR using the NO spin trap, Fe-MGD. Attenuated ROS in ecSOD Tg myocytes was also supported by decreased production of peroxynitrite (ONOO−). Increased NO bioavailability was confirmed by attenuated guanylate cyclase-dependent (p-VASP) signaling. In conclusion, attenuation of ROS levels by cardiac-specific ecSOD overexpression increases NO bioavailability in response to ischemia/reperfusion and protects against reperfusion injury. These findings are the first to demonstrate increased NO bioavailability with attenuation of ROS by direct measurement of these reactive species (EPR, reactive fluorescent dyes) with cardiac-specific ecSOD expression. This is also the first indication that the predominantly extracellular SOD isoform is capable of cytosolic localization that affects myocardial intracellular signal transduction and function.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-012-0305-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3505528  PMID: 23099819
NO-bioavailability; Extracellular superoxide dismutase; Cardioprotection; Ischemia/reperfusion injury; Peroxynitrite
16.  Remote Effects of Lower Limb Ischemia-Reperfusion: Impaired Lung, Unchanged Liver, and Stimulated Kidney Oxidative Capacities 
BioMed Research International  2014;2014:392390.
Remote organ impairments are frequent and increase patient morbidity and mortality after lower limb ischemia-reperfusion (IR). We challenged the hypothesis that lower limb IR might also impair lung, renal, and liver mitochondrial respiration. Two-hour tourniquet-induced ischemia was performed on both hindlimbs, followed by a two-hour reperfusion period in C57BL6 mice. Lungs, liver and kidneys maximal mitochondrial respiration (Vmax⁡), complexes II, III, and IV activity (Vsucc), and complex IV activity (VTMPD) were analyzed on isolated mitochondria. Lower limb IR decreased significantly lung Vmax⁡ (29.4 ± 3.3 versus 24 ± 3.7 μmol O2/min/g dry weight, resp.; P = 0.042) and tended to reduce Vsucc and VTMPD. IR did not modify liver but increased kidneys mitochondrial respiration (79.5 ± 19.9 versus 108.6 ± 21.4, P = 0.035, and 126 ± 13.4 versus 142.4 ± 10.4 μmol O2/min/g dry weight for Vmax⁡ and Vsucc, resp.). Kidneys mitochondrial coupling was increased after IR (6.5 ± 1.3 versus 8.8 ± 1.1, P = 0.008). There were no histological changes in liver and kidneys. Thus, lung mitochondrial dysfunction appears as a new early marker of hindlimb IR injuries in mice. Further studies will be useful to determine whether enhanced kidneys mitochondrial function allows postponing kidney impairment in lower limb IR setting.
PMCID: PMC4142554  PMID: 25180180
17.  Remote ischemic preconditioning confers late protection against myocardial ischemia-reperfusion injury in mice by upregulating interleukin-10 
Basic research in cardiology  2012;107(4):277.
Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia-reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of interleukin (IL)-10. Mice were exposed to lower limb RIPC or sham ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120). These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies. In IL-10 knockout mice, RIPC cardioprotection was lost, but it was mimicked by exogenous IL-10. Administration of IL-10 to isolated perfused hearts increased phosphory-lation of the protein kinase Akt and limited infarct size after I-30/R-120. In wild-type mice, RIPC increased plasma and cardiac IL-10 protein levels and caused activation of Akt and endothelial nitric oxide synthase in the heart at 24 h, which was also blocked by anti-IL-10 receptor antibodies. In the gastrocnemius muscle, RIPC resulted in immediate inactivation of the phosphatase PTEN and activation of Stat3, with increased IL-10 expression 24 h later. Myocyte-specific PTEN inactivation led to increased Stat3 phosphorylation and IL-10 protein expression in the gastrocnemius muscle. Taken together, these results suggest that RIPC induces late protection against myocardial IR injury by increasing expression of IL-10 in the remote muscle, followed by release of IL-10 into the circulation, and activation of protective signaling pathways in the heart. This study provides a scientific basis for the use of RIPC to confer systemic protection against IR injury.
PMCID: PMC3596418  PMID: 22752341
Remote ischemic preconditioning; Interleukin-10; Reperfusion injury; Phosphatase and tensin homologue deleted on chromosome ten; Stat3
18.  Endoplasmic reticulum stress activation during total knee arthroplasty 
Physiological Reports  2013;1(3):e00052.
Total knee arthroplasty (TKA) is the most common remediation for knee pain from osteoarthritis (OA) and is performed 650,000 annually in the U.S. A tourniquet is commonly used during TKA which causes ischemia and reperfusion (I/R) to the lower limb but the effects of I/R on muscle are not fully understood. Previous reports suggest upregulation of cell stress and catabolism and downregulation of markers of cap-dependent translation during and after TKA. I/R has also been shown to cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). We hypothesized that the UPR would be activated in response to ER stress during TKA. We obtained muscle biopsies from the vastus lateralis at baseline, before TKA; at maximal ischemia, prior to tourniquet deflation; and during reperfusion in the operating room. Phosphorylation of 4E-BP1 and AKT decreased during ischemia (−28%, P < 0.05; −20%, P < 0.05, respectively) along with an increase in eIF2α phosphorylation (64%, P < 0.05) suggesting decreased translation initiation. Cleaved ATF6 protein increased in ischemia (39%, P = 0.056) but returned to baseline during reperfusion. CASP3 activation increased during reperfusion compared to baseline (23%, P < 0.05). XBP1 splicing assays revealed an increase in spliced transcript during ischemia (31%, P < 0.05) which diminished during reperfusion. These results suggest that in response to I/R during TKA all three branches of the ER stress response are activated.
PMCID: PMC3804266  PMID: 24159375
Clinical; ER stress; ischemia reperfusion; muscle; unfolded protein response
19.  Endoplasmic Reticulum Stress Activation during Total Knee Arthroplasty 
Physiological reports  2013;1(3):e00052-.
Total knee arthroplasty (TKA) is the most common remediation for knee pain from osteoarthritis (OA) and is performed 650,000 annually in the U.S. A tourniquet is commonly used during TKA which causes ischemia and reperfusion (I/R) to the lower limb but the effects of I/R on muscle are not fully understood. Previous reports suggest upregulation of cell-stress and catabolism and downregulation of markers of cap-dependent translation during and after TKA. I/R has also been shown to cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). We hypothesized that the UPR would be activated in response to ER stress during TKA. We obtained muscle biopsies from the vastus lateralis at baseline, before TKA; at maximal ischemia, prior to tourniquet deflation; and during reperfusion in the operating room. Phosphorylation of 4E-BP1 and AKT decreased during ischemia (−28%, p < .05; −20%, p < .05 respectively) along with an increase in eIF2α phosphorylation (64%, p < .05) suggesting decreased translation initiation. Cleaved ATF6 protein increased in ischemia (39%, p = .056) but returned to baseline during reperfusion. CASP3 activation increased during reperfusion compared to baseline (23%, p < .05). XBP1 splicing assays revealed an increase in spliced transcript during ischemia (31%, p < .05) which diminished during reperfusion. These results suggest that in response to I/R during TKA all three branches of the ER stress response are activated.
PMCID: PMC3804266  PMID: 24159375
Clinical; Ischemia Reperfusion; ER stress; Unfolded Protein Response; Muscle
20.  Partial hexokinase II knockout results in acute ischemia–reperfusion damage in skeletal muscle of male, but not female, mice 
Pflugers Archiv   2010;459(5):705-712.
Cellular studies have demonstrated a protective role of mitochondrial hexokinase against oxidative insults. It is unknown whether HK protective effects translate to the in vivo condition. In the present study, we hypothesize that HK affects acute ischemia–reperfusion injury in skeletal muscle of the intact animal. Male and female heterozygote knockout HKII (HK+/-), heterozygote overexpressed HKII (HKtg), and their wild-type (WT) C57Bl/6 littermates mice were examined. In anesthetized animals, the left gastrocnemius medialis (GM) muscle was connected to a force transducer and continuously stimulated (1-Hz twitches) during 60 min ischemia and 90 min reperfusion. Cell survival (%LDH) was defined by the amount of cytosolic lactate dehydrogenase (LDH) activity still present in the reperfused GM relative to the contralateral (non-ischemic) GM. Mitochondrial HK activity was 72.6 ± 7.5, 15.7 ± 1.7, and 8.8 ± 0.9 mU/mg protein in male mice, and 72.7 ± 3.7, 11.2 ± 1.4, and 5.9 ± 1.1 mU/mg in female mice for HKtg, WT, and HK+/-, respectively. Tetanic force recovery amounted to 33 ± 7% for male and 17 ± 4% for female mice and was similar for HKtg, WT, and HK+/-. However, cell survival was decreased (p = 0.014) in male HK+/- (82 ± 4%LDH) as compared with WT (98 ± 5%LDH) and HKtg (97 ± 4%LDH). No effects of HKII on cell survival was observed in female mice (92 ± 2% LDH). In conclusion, in this mild model of acute in vivo ischemia–reperfusion injury, a partial knockout of HKII was associated with increased cell death in male mice. The data suggest for the first time that HKII mediates skeletal muscle ischemia–reperfusion injury in the intact male animal.
PMCID: PMC2842566  PMID: 20182739
Mitochondria; Cell death; Ischemia; Muscle; Muscle ischemia
21.  How effective are alprostadil and hydrocortisone on reperfusion injury in kidney after distant organ ischemia? 
After reestablishment of blood flow to ischemic limb recirculation of free radicals may cause ischemia-reperfusion injury in many organs. This study designed to investigate effects of hydrocortisone and alprostadil distant injury to kidneys by both measuring biochemical markers of oxidative stress and histopathologic examination in an experimental rat model of hind limb ischemia-reperfusion.
Materials and Methods:
This study conducted in Isfahan University of Medical Sciences during 2011–2012. Ischemia was established by infra renal aortic clamping for 60 min in 32 male Wistar rats. Animals were divided into those receiving alprostadil (group ischemia-reperfusion plus alprostadil (IR/A), n = 8), those receiving hydrocortisone (group ischemia-reperfusion plus hydrocortisone (IR/H), n = 8), control group (group ischemia-reperfusion (IR), n = 8), and sham group (n = 8). After 120 min of reperfusion both kidneys were removed. Levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) as indirect markers of oxidative injury was measured. Finally all data in different groups were compared using the analysis of variance (ANOVA) test by Statistical Package for Social Sciences (SPSS) version 16.
Administration of alprostadil or hydrocortisone does not improve the biochemical parameters of oxidative injury including MDA and SOD. However, statistically significant difference was seen in GSH level among sham and IR groups. Mean (± standard deviation (SD)) concentration of GSH in IR, IR/A, IR/H, and sham groups were 1028.77 (72.65), 924.82 (70.66), 1000.28 (108.77), and 846.69 (163.52), respectively (P = 0.015). Histopathological study of specimens did not show any significant changes between groups.
Alprostadil and hydrocortisone do not improve the kidney GSH, SOD, and MDA level and kidney releases its GSH reserve during ischemia-reperfusion event, and another point is that, 3 h of ischemia-reperfusion does not develop injury in kidney.
PMCID: PMC3872582  PMID: 24381617
Alprostadil; hydrocortisone; ischemia-reperfusion injury; oxidative stress
22.  Protective effect of edaravone for tourniquet-induced ischemia-reperfusion injury on skeletal muscle in murine hindlimb 
Studies have shown that ischemia-reperfusion (I/R) produces free radicals leading to lipid peroxidation and damage to skeletal muscle. The purposes of this study were 1) to assess the histological findings of gastrocnemius muscle (GC) and tibialis anterior muscle (TA) in I/R injury model mice, 2) to histologically analyze whether a single pretreatment of edaravone inhibits I/R injury to skeletal muscle in murine models and 3) to evaluate the effect of oxidative stress on these muscles.
C57BL6 mice were divided in two groups, with one group receiving 3 mg/kg intraperitoneal injections of edaravone (I/R + Ed group) and the other group receiving an identical amount of saline (I/R group) 30 minutes before ischemia. Edaravone (3-methy-1-pheny1-2-pyrazolin-5-one) is a potent and novel synthetic scavenger of free radicals. This drug inhibits both nonenzymatic lipid peroxidation and the lipoxygenase pathway, in addition to having potent antioxidant effects against ischemia reperfusion. The duration of the ischemia was 1.5 hours, with reperfusion at either 24 or 72 hours (3 days). Specimens of gastrocnemius (GC) and anterior tibialis (TA) were removed for histological evaluation and biochemical analysis.
This model of I/R injury was highly reproducible in histologic muscle damage. In the histologic damage score, the mean muscle fibers and inflammatory cell infiltration in the I/R + Ed group were significantly less than the corresponding values of observed in the I/R group. Thus, pretreatment with edaravone was observed to have a protective effect on muscle damage after a period of I/R in mice. In addition, the mean muscle injury score in the I/R + Ed group was also significantly less than the I/R group. In the I/R + Ed group, the mean malondialdehyde (MDA) level was lower than in the I/R group and western-blotting revealed that edaravone pretreatment decreased the level of inducible nitric oxide synthase (iNOS) expression.
Edaravone was found to have a protective effect against I/R injury by directly inhibiting lipid peroxidation of the myocyte by free radicals in skeletal muscles and may also reduce the secondary edema and inflammatory infiltration incidence of oxidative stress on tissue.
PMCID: PMC3614524  PMID: 23530927
Ischemia-reperfusion injury; Skeletal muscle; Free radical scavenger; Edaravone; iNOS
23.  Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66Shc to Preserve Cardiac Function after Ischemia 
PLoS ONE  2014;9(12):e113534.
Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66Shc protects against IR injury, but it is unknown if and when p66Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66Shc activation during IR is associated with cardioprotection.
Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCβ inhibitor. Phosphorylation of p66Shc at serine 36 and levels of p66Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion.
Phosphorylation of p66Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion.
Our results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.
PMCID: PMC4250075  PMID: 25436907
24.  Formation of Hydrogen Peroxide and Reduction of Peroxynitrite via Dismutation of Superoxide at Reperfusion Enhances Myocardial Blood Flow and Oxygen Consumption in Postischemic Mouse Heart 
Reactive oxygen/nitrogen species suppress myocardial oxygen consumption. In this study, we determined that endogenous hydrogen peroxide through dismutation of superoxide enhances postischemic myocardial blood perfusion and oxygen consumption. Electron paramagnetic resonance oximetry was applied to monitor in vivo tissue Po2 in mouse heart subjected to regional ischemia reperfusion. Heart rate, arterial blood pressure, blood flow, infarction, and activities of mitochondrial NADH dehydrogenase and cytochrome c oxidase were measured in six groups of wild-type (WT) and endothelial nitric-oxide synthase knock-out (eNOS−/−) mice treated with phosphate-buffered saline (PBS), superoxide dismutase mimetic (SODm) M40403 [a manganese(II)-bis(cyclohexylpyridine)-substituted macrocyclic superoxide dismutase mimetic, C21H35Cl2MnN5], 10006329 EUK 134 [EUK134, manganese 3-methoxy N,N1-bis(salicyclidene)ethylenediamine chloride], and SODm plus glibenclamide to study the protective effect of hydrogen peroxide via dismutation of superoxide on the activation of sarcolemmal potassium channels. In the PBS group, there was an overshoot of tissue Po2 after reperfusion. Treatment with SODm, EUK134, and SODm + glibenclamide protected mitochondrial enzyme activities, reduced infarct size, and suppressed the post-ischemic hyperoxygenation. In particular, in the SODm-treated group, there was a transient peak of tissue Po2 at 9 min after reperfusion, which was dependent on endogenous hydrogen peroxide but not nitric oxide formation as it appeared in both WT and eNOS−/− mice. Blood flow and rate pressure product were higher in the SODm group than in other groups, which contributed to the transient oxygen peak. Thus, SOD mimetics protected mouse heart from superoxide-induced reperfusion injury. With treatment of different SOD mimetics, it is concluded that endogenous hydrogen peroxide via dismutation of superoxide at reperfusion enhances postischemic myocardial blood perfusion and mitochondrial oxygen consumption, possibly through activation of sarcolemmal ATP-sensitive potassium channels.
PMCID: PMC2615247  PMID: 18685120
25.  Ischemia/Reperfusion Injury Protection by Mesenchymal Stem Cell Derived Antioxidant Capacity 
Stem Cells and Development  2013;22(18):2497-2507.
Mesenchymal stem cell (MSC) transplantation after ischemia/reperfusion (I/R) injury reduces infarct size and improves cardiac function. We used mouse ventricular myocytes (VMs) in an in vitro model of I/R to determine the mechanism by which MSCs prevent reperfusion injury by paracrine signaling. Exposure of mouse VMs to an ischemic challenge depolarized their mitochondrial membrane potential (Ψmito), increased their diastolic Ca2+, and significantly attenuated cell shortening. Reperfusion of VMs with Ctrl tyrode or MSC-conditioned tyrode (ConT) resulted in a transient increase of the Ca2+ transient amplitudes in all cells. ConT-reperfused cells exhibited a decreased number early after depolarization (EADs) (ConT: 6.3% vs. Ctrl: 28.4%) and prolonged survival (ConT: 58% vs. Ctrl: 33%). Ψmito rapidly recovered in Ctrl as well as ConT-treated VMs on reperfusion; however, in Ctrl solution, an exaggerated hyperpolarization of Ψmito was determined that preceded the collapse of Ψmito. The ability of ConT to attenuate the hyperpolarization of Ψmito was suppressed on inhibition of the PI3K/Akt signaling pathway or IK,ATP. However, protection of Ψmito was best mimicked by the reactive oxygen species (ROS) scavenger mitoTEMPO. Analysis of ConT revealed a significant antioxidant capacity that was linked to the presence of extracellular superoxide dismutase (SOD3) in ConT. In conclusion, MSC ConT protects VMs from simulated I/R injury by its SOD3-mediated antioxidant capacity and by delaying the recovery of Ψmito through Akt-mediated opening of IK,ATP. These changes attenuate reperfusion-induced ROS production and prevent the opening of the permeability transition pore and arrhythmic Ca2+ release.
PMCID: PMC3760058  PMID: 23614555

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