Publications by authors named "Xiangru Lu"

54 Publications

Myocardium-Specific Deletion of Rac1 Causes Ventricular Noncompaction and Outflow Tract Defects.

J Cardiovasc Dev Dis 2021 Mar 15;8(3). Epub 2021 Mar 15.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada.

Background: Left ventricular noncompaction (LVNC) is a cardiomyopathy that can lead to arrhythmias, embolic events and heart failure. Despite our current knowledge of cardiac development, the mechanisms underlying noncompaction of the ventricular myocardium are still poorly understood. The small GTPase acts as a crucial regulator of numerous developmental events. The present study aimed to investigate the cardiomyocyte specific role of in embryonic heart development.

Methods And Results: The transgenic mice were crossed with mice to generate mice with a cardiomyocyte specific deletion of () during heart development. Embryonic hearts at E12.5-E18.5 were collected for histological analysis. Overall, hearts displayed a bifid apex, along with hypertrabeculation and a thin compact myocardium. hearts also exhibited ventricular septal defects (VSDs) and double outlet right ventricle (DORV) or overriding aorta. Cardiomyocytes had a rounded morphology and were highly disorganized, and the myocardial expression of Scrib, a planar cell polarity protein, was reduced in hearts. In addition, cell proliferation rate was significantly decreased in the ventricular myocardium at E9.5.

Conclusions: deficiency in the myocardium impairs cardiomyocyte elongation and organization, and proliferative growth of the heart. A spectrum of CHDs arises in hearts, implicating signaling in the ventricular myocardium as a crucial regulator of OFT alignment, along with compact myocardium growth and development.
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http://dx.doi.org/10.3390/jcdd8030029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001666PMC
March 2021

NOX2 Is Critical to Endocardial to Mesenchymal Transition and Heart Development.

Oxid Med Cell Longev 2020 16;2020:1679045. Epub 2020 Jun 16.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Canada.

NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, deficiency disrupted EndMT of atrioventricular cushion explants . Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of was associated with reduced expression of , , , , and , which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.
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http://dx.doi.org/10.1155/2020/1679045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320281PMC
May 2021

Sapropterin reduces coronary artery malformation in offspring of pregestational diabetes mice.

Nitric Oxide 2020 01 7;94:9-18. Epub 2019 Oct 7.

Department of Physiology and Pharmacology, London, Ontario, Canada; Department of Medicine, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; Children's Health Research Institute, London, Ontario, Canada. Electronic address:

Endothelial nitric oxide synthase (eNOS) and oxidative stress are critical to embryonic coronary artery development. Maternal diabetes increases oxidative stress and reduces eNOS activity in the fetal heart. Sapropterin (Kuvan®) is an orally active, synthetic form of tetrahydrobiopterin (BH4) and a co-factor for eNOS with antioxidant properties. The aim of the present study was to examine the effects of sapropterin on fetal coronary artery development during pregestational diabetes in mice. Diabetes was induced by streptozotocin to adult female C57BL/6 mice. Sapropterin (10 mg/kg/day) was orally administered to pregnant mice from E0.5 to E18.5. Fetal hearts were collected at E18.5 for coronary artery morphological analysis. Sapropterin treatment to diabetic dams reduced the incidence of coronary artery malformation in offspring from 50.0% to 20.6%. Decreases in coronary artery luminal diameter, volume and abundance in fetal hearts from diabetic mothers, were prevented by sapropterin treatment. Maternal diabetes reduced epicardial epithelial-to-mesenchymal transition (EMT) and expression of transcription and growth factors critical to coronary artery development including hypoxia-inducible factor 1a (Hif1a), Snail1, Slug, β-catenin, retinaldehyde dehydrogenase 2 (Aldh1a2), basic fibroblast growth factor (bFGF) and vascular endothelial group factor receptor 2 (Vegfr2) in E12.5 hearts. Additionally, eNOS phosphorylation was lower while oxidative stress was higher in E12.5 hearts from maternal diabetes. Notably, these abnormalities were all restored to normal levels after sapropterin treatment. In conclusion, sapropterin treatment increases eNOS activity, lowers oxidative stress and reduces coronary artery malformation in offspring of pregestational diabetes. Sapropterin may have therapeutic potential in preventing coronary artery malformation in maternal diabetes.
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http://dx.doi.org/10.1016/j.niox.2019.10.002DOI Listing
January 2020

Maternal voluntary exercise mitigates oxidative stress and incidence of congenital heart defects in pre-gestational diabetes.

J Cell Mol Med 2019 08 18;23(8):5553-5565. Epub 2019 Jun 18.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, Children's Health Research Institute, London, ON, Canada.

Women with pre-gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia-induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre-gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes-induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial-mesenchymal transition at E12.5 was significantly lower with pre-gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non-exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre-gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.
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http://dx.doi.org/10.1111/jcmm.14439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6653048PMC
August 2019

Sapropterin Treatment Prevents Congenital Heart Defects Induced by Pregestational Diabetes Mellitus in Mice.

J Am Heart Assoc 2018 11;7(21):e009624

1 Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London Ontario Canada.

Background Tetrahydrobiopterin is a cofactor of endothelial NO synthase ( eNOS ), which is critical to embryonic heart development. We aimed to study the effects of sapropterin (Kuvan), an orally active synthetic form of tetrahydrobiopterin on eNOS uncoupling and congenital heart defects ( CHD s) induced by pregestational diabetes mellitus in mice. Methods and Results Adult female mice were induced to pregestational diabetes mellitus by streptozotocin and bred with normal male mice to produce offspring. Pregnant mice were treated with sapropterin or vehicle during gestation. CHD s were identified by histological analysis. Cell proliferation, eNOS dimerization, and reactive oxygen species production were assessed in the fetal heart. Pregestational diabetes mellitus results in a spectrum of CHD s in their offspring. Oral treatment with sapropterin in the diabetic dams significantly decreased the incidence of CHD s from 59% to 27%, and major abnormalities, such as atrioventricular septal defect and double-outlet right ventricle, were absent in the sapropterin-treated group. Lineage tracing reveals that pregestational diabetes mellitus results in decreased commitment of second heart field progenitors to the outflow tract, endocardial cushions, and ventricular myocardium of the fetal heart. Notably, decreased cell proliferation and cardiac transcription factor expression induced by maternal diabetes mellitus were normalized with sapropterin treatment. Furthermore, sapropterin administration in the diabetic dams increased eNOS dimerization and lowered reactive oxygen species levels in the fetal heart. Conclusions Sapropterin treatment in the diabetic mothers improves eNOS coupling, increases cell proliferation, and prevents the development of CHD s in the offspring. Thus, sapropterin may have therapeutic potential in preventing CHD s in pregestational diabetes mellitus.
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http://dx.doi.org/10.1161/JAHA.118.009624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404194PMC
November 2018

S-Nitrosylation of STIM1 by Neuronal Nitric Oxide Synthase Inhibits Store-Operated Ca Entry.

J Mol Biol 2018 06 27;430(12):1773-1785. Epub 2018 Apr 27.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada; Department of Medicine, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada. Electronic address:

Store-operated Ca entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca release-activated Ca channel current (I) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and I in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca depletion-dependent oligomerization of the luminal Ca-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.
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http://dx.doi.org/10.1016/j.jmb.2018.04.028DOI Listing
June 2018

A charge-sensing region in the stromal interaction molecule 1 luminal domain confers stabilization-mediated inhibition of SOCE in response to -nitrosylation.

J Biol Chem 2018 06 16;293(23):8900-8911. Epub 2018 Apr 16.

From the Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada

Store-operated Ca entry (SOCE) is a major Ca signaling pathway facilitating extracellular Ca influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca stores. Stromal interaction molecule 1 (STIM1) is the Ca sensor that activates SOCE following ER/SR Ca depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca concentrations. Low ER Ca levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca channels on the plasma membrane. NO-mediated nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that nitrosylation of Cys and Cys in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca depletion-dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys and Cysnitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.
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http://dx.doi.org/10.1074/jbc.RA117.000503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995509PMC
June 2018

Maternal diabetes up-regulates NOX2 and enhances myocardial ischaemia/reperfusion injury in adult offspring.

J Cell Mol Med 2018 04 29;22(4):2200-2209. Epub 2018 Jan 29.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up-regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose-tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF-Akt (protein kinase B)-mammalian target of rapamycin (mTOR)-NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose-induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF-Akt-mTOR-NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.
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http://dx.doi.org/10.1111/jcmm.13500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867143PMC
April 2018

Cardiomyocyte specific overexpression of a 37 amino acid domain of regulator of G protein signalling 2 inhibits cardiac hypertrophy and improves function in response to pressure overload in mice.

J Mol Cell Cardiol 2017 07 19;108:194-202. Epub 2017 Jun 19.

Department of Physiology and Pharmacology, University of Western Ontario, London, ON, N6A5C1, Canada. Electronic address:

Regulator of G protein signalling 2 (RGS2) is known to play a protective role in maladaptive cardiac hypertrophy and heart failure via its ability to inhibit G- and G- mediated GPCR signalling. We previously demonstrated that RGS2 can also inhibit protein translation and can thereby attenuate cell growth. This G protein-independent inhibitory effect has been mapped to a 37 amino acid domain (RGS2) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed in neonatal rat cardiomyocytes, RGS2 attenuates both protein synthesis and hypertrophy induced by G- and G- activating agents. In the current study, we investigated the potential cardioprotective role of RGS2 by determining whether RGS2 transgenic (RGS2 TG) mice with cardiomyocyte specific overexpression of RGS2 show resistance to the development of hypertrophy in comparison to wild-type (WT) controls. Using transverse aortic constriction (TAC) in a pressure-overload hypertrophy model, we demonstrated that cardiac hypertrophy was inhibited in RGS2 TG mice compared to WT controls following four weeks of TAC. Expression of the hypertrophic markers atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC-β) was also reduced in RGS2 TG compared to WT TAC animals. Furthermore, cardiac function in RGS2 TG TAC mice was significantly improved compared to WT TAC mice. Notably, cardiomyocyte cell size was significantly decreased in TG compared to WT TAC mice. These results suggest that RGS2 may limit pathological cardiac hypertrophy at least in part via the function of its eIF2B-binding domain.
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http://dx.doi.org/10.1016/j.yjmcc.2017.06.007DOI Listing
July 2017

North American ginseng inhibits myocardial NOX2-ERK1/2 signaling and tumor necrosis factor-α expression in endotoxemia.

Pharmacol Res 2016 09 16;111:217-225. Epub 2016 Jun 16.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada. Electronic address:

Sepsis is a systemic inflammatory response to infection with a high mortality but has no specific treatment despite decades of research. North American (NA) ginseng (Panax quinquefolius) is a popular natural health product with anti-oxidant and anti-inflammatory properties. The aim of the present study was to investigate the effects of NA ginseng on pro-inflammatory cytokine expression and cardiac function in endotoxemia, a model of sepsis. Mice were challenged with lipopolysaccharide (LPS) to induce endotoxemia. Myocardial expression of tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory cytokine that causes cardiac dysfunction, was upregulated in mice with endotoxemia, which was accompanied by increases in NOX2 expression, superoxide generation and ERK1/2 phosphorylation. Notably, pretreatment with NA ginseng aqueous extract (50mg/kg/day, oral gavage) for 5days significantly inhibited NOX2 expression, superoxide generation, ERK1/2 phosphorylation and TNF-α expression in the heart during endotoxemia. Importantly, cardiac function and survival in endotoxemic mice were significantly improved. Additionally, pretreatment with ginseng extract inhibited superoxide generation, ERK1/2 phosphorylation and TNF-α expression induced by LPS in cultured cardiomyocytes. We conclude that NA ginseng inhibits myocardial NOX2-ERK1/2-TNF-α signaling pathway and improves cardiac function in endotoxemia, suggesting that NA ginseng may have the potential in the prevention of clinical sepsis.
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http://dx.doi.org/10.1016/j.phrs.2016.06.010DOI Listing
September 2016

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration.

J Vis Exp 2016 05 24(111). Epub 2016 May 24.

Department of Physiology and Pharmacology, Western University;

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.
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http://dx.doi.org/10.3791/54100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927718PMC
May 2016

Inhibition of Rac1 reduces store overload-induced calcium release and protects against ventricular arrhythmia.

J Cell Mol Med 2016 08 25;20(8):1513-22. Epub 2016 May 25.

Department of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada.

Rac1 is a small GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload-induced Ca(2+) release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1(f/f) and cardiac-specific Rac1 knockdown (Rac1(ckd) ) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1(f/f) mice. Remarkably, I/R-induced ventricular arrhythmia was significantly decreased in Rac1(ckd) compared to Rac1(f/f) mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R-induced ventricular arrhythmia. Ca(2+) imaging analysis showed that in response to a 6 mM external Ca(2+) concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca(2+) waves in Rac1(f/f) cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R-induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1(ckd) mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease.
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http://dx.doi.org/10.1111/jcmm.12840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956946PMC
August 2016

Rac1 Signaling Is Required for Anterior Second Heart Field Cellular Organization and Cardiac Outflow Tract Development.

J Am Heart Assoc 2015 Dec 31;5(1). Epub 2015 Dec 31.

Departments of Physiology and Pharmacology, Medicine and Pediatrics, Schulich School of Medicine and Dentistry, Collaborative Program in Developmental Biology, Children's Health Research Institute, University of Western Ontario, London, Ontario, Canada (C.L., Y.L., X.L., M.K., T.A.D., Q.F.).

Background: The small GTPase Rac1 regulates diverse cellular functions, including both apicobasal and planar cell polarity pathways; however, its role in cardiac outflow tract (OFT) development remains unknown. In the present study, we aimed to examine the role of Rac1 in the anterior second heart field (SHF) splanchnic mesoderm and subsequent OFT development during heart morphogenesis.

Methods And Results: Using the Cre/loxP system, mice with an anterior SHF-specific deletion of Rac1 (Rac1(SHF)) were generated. Embryos were collected at various developmental time points for immunostaining and histological analysis. Intrauterine echocardiography was also performed to assess aortic valve blood flow in embryos at embryonic day 18.5. The Rac1(SHF) splanchnic mesoderm exhibited disruptions in SHF progenitor cellular organization and proliferation. Consequently, this led to a spectrum of OFT defects along with aortic valve defects in Rac1(SHF) embryos. Mechanistically, it was found that the ability of the Rac1(SHF) OFT myocardial cells to migrate into the proximal OFT cushion was severely reduced. In addition, expression of the neural crest chemoattractant semaphorin 3c was decreased. Lineage tracing showed that anterior SHF contribution to the OFT myocardium and aortic valves was deficient in Rac1(SHF) hearts. Furthermore, functional analysis with intrauterine echocardiography at embryonic day 18.5 showed aortic valve regurgitation in Rac1(SHF) hearts, which was not seen in control hearts.

Conclusions: Disruptions of Rac1 signaling in the anterior SHF results in aberrant progenitor cellular organization and defects in OFT development. Our data show Rac1 signaling to be a critical regulator of cardiac OFT formation during embryonic heart development.
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http://dx.doi.org/10.1161/JAHA.115.002508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859369PMC
December 2015

Pregestational diabetes induces fetal coronary artery malformation via reactive oxygen species signaling.

Diabetes 2015 Apr 24;64(4):1431-43. Epub 2014 Nov 24.

Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada Lawson Health Research Institute, London Health Sciences Centre, London, Ontario, Canada Department of Medicine, University of Western Ontario, London, Ontario, Canada

Hypoplastic coronary artery disease is a congenital coronary artery malformation associated with a high risk of sudden cardiac death. However, the etiology and pathogenesis of hypoplastic coronary artery disease remain undefined. Pregestational diabetes increases reactive oxygen species (ROS) levels and the risk of congenital heart defects. We show that pregestational diabetes in mice induced by streptozotocin significantly increased 4-hydroxynonenal production and decreased coronary artery volume in fetal hearts. Pregestational diabetes also impaired epicardial epithelial-to-mesenchymal transition (EMT) as shown by analyses of the epicardium, epicardial-derived cells, and fate mapping. Additionally, the expression of hypoxia-inducible factor 1α (Hif-1α), Snail1, Slug, basic fibroblast growth factor (bFgf), and retinaldehyde dehydrogenase (Aldh1a2) was decreased and E-cadherin expression was increased in the hearts of fetuses of diabetic mothers. Of note, these abnormalities were all rescued by treatment with N-acetylcysteine (NAC) in diabetic females during gestation. Ex vivo analysis showed that high glucose levels inhibited epicardial EMT, which was reversed by NAC treatment. We conclude that pregestational diabetes in mice can cause coronary artery malformation through ROS signaling. This study may provide a rationale for further clinical studies to investigate whether pregestational diabetes could cause hypoplastic coronary artery disease in humans.
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http://dx.doi.org/10.2337/db14-0190DOI Listing
April 2015

Rac1 signaling is critical to cardiomyocyte polarity and embryonic heart development.

J Am Heart Assoc 2014 Oct 14;3(5):e001271. Epub 2014 Oct 14.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada (C.L., X.L., Q.F.) Department of Medicine, Schulich School of Medicine and Dentistry The University of Western Ontario, London, Ontario, Canada (Q.F.) Lawson Health Research Institute, The University of Western Ontario, London, Ontario, Canada (M.L., Q.F.).

Background: Defects in cardiac septation are the most common form of congenital heart disease, but the mechanisms underlying these defects are still poorly understood. The small GTPase Rac1 is implicated in planar cell polarity of epithelial cells in Drosophila; however, its role in mammalian cardiomyocyte polarity is not clear. We tested the hypothesis that Rac1 signaling in the second heart field regulates cardiomyocyte polarity, chamber septation, and right ventricle development during embryonic heart development.

Methods And Results: Mice with second heart field-specific deficiency of Rac1 (Rac1(SHF)) exhibited ventricular and atrial septal defects, a thinner right ventricle myocardium, and a bifid cardiac apex. Fate-mapping analysis showed that second heart field contribution to the interventricular septum and right ventricle was deficient in Rac1(SHF) hearts. Notably, cardiomyocytes had a spherical shape with disrupted F-actin filaments in Rac1(SHF) compared with elongated and well-aligned cardiomyocytes in littermate controls. Expression of Scrib, a core protein in planar cell polarity, was lost in Rac1(SHF) hearts with decreased expression of WAVE and Arp2/3, leading to decreased migratory ability. In addition, Rac1-deficient neonatal cardiomyocytes displayed defects in cell projections, lamellipodia formation, and cell elongation. Furthermore, apoptosis was increased and the expression of Gata4, Tbx5, Nkx2.5, and Hand2 transcription factors was decreased in the Rac1(SHF) right ventricle myocardium.

Conclusions: Deficiency of Rac1 in the second heart field impairs elongation and cytoskeleton organization of cardiomyocytes and results in congenital septal defects, thin right ventricle myocardium, and a bifid cardiac apex. Our study suggests that Rac1 signaling is critical to cardiomyocyte polarity and embryonic heart development.
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http://dx.doi.org/10.1161/JAHA.114.001271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323834PMC
October 2014

Cardiac-specific overexpression of human stem cell factor promotes epicardial activation and arteriogenesis after myocardial infarction.

Circ Heart Fail 2014 Sep 8;7(5):831-42. Epub 2014 Aug 8.

From the Departments of Physiology and Pharmacology (F.-L.X., Y.L., X.L., D.L.J., Q.F.) and Medicine (D.L.J., Q.F.), and Lawson Health Research Institute (D.L.J., Q.F.), Western University, London, Ontario, Canada.

Background: The adult epicardium is a potential source of cardiac progenitors after myocardial infarction (MI). We tested the hypothesis that cardiomyocyte-specific overexpression of membrane-associated human stem cell factor (hSCF) enhances epicardial activation, epicardium-derived cells (EPDCs) production, and myocardial arteriogenesis post MI.

Methods And Results: Wild-type and the inducible cardiac-specific hSCF transgenic (hSCF/tetracycline transactivator) mice were subjected to MI. Wilms tumor-1 (Wt1)-positive epicardial cells were higher in hSCF/tetracycline transactivator compared with wild-type mice 3 days post MI. Arteriole density was significantly higher in the peri-infarct area of hSCF/tetracycline transactivator mice compared with wild-type mice 5 days post MI. In cultured EPDCs, adenoviral hSCF treatment significantly increased cell proliferation and growth factor expression. Furthermore, adenoviral hSCF treatment in wild-type cardiomyocytes significantly increased EPDC migration. These effects of hSCF overexpression on EPDC proliferation and growth factor expression were all abrogated by ACK2, a neutralizing antibody against c-kit. Finally, lineage tracing using ROSA(mTmG);Wt1(CreER) mice showed that adenoviral hSCF treatment increased Wt1(+) lineage-derived EPDC migration into the infarcted myocardium 5 days post MI, which was inhibited by ACK2.

Conclusions: Cardiomyocyte-specific overexpression of hSCF promotes epicardial activation and myocardial arteriogenesis post MI.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.114.001423DOI Listing
September 2014

N-Acetylcysteine prevents congenital heart defects induced by pregestational diabetes.

Cardiovasc Diabetol 2014 Feb 18;13:46. Epub 2014 Feb 18.

Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada.

Background: Pregestational diabetes is a major risk factor of congenital heart defects (CHDs). Glutathione is depleted and reactive oxygen species (ROS) production is elevated in diabetes. In the present study, we aimed to examine whether treatment with N-acetylcysteine (NAC), which increases glutathione synthesis and inhibits ROS production, prevents CHDs induced by pregestational diabetes.

Methods: Female mice were treated with streptozotocin (STZ) to induce pregestational diabetes prior to breeding with normal males to produce offspring. Some diabetic mice were treated with N-acetylcysteine (NAC) in drinking water from E0.5 to the end of gestation or harvesting of the embryos. CHDs were identified by histology. ROS levels, cell proliferation and gene expression in the fetal heart were analyzed.

Results: Our data show that pregestational diabetes resulted in CHDs in 58% of the offspring, including ventricular septal defect (VSD), atrial septal defect (ASD), atrioventricular septal defects (AVSD), transposition of great arteries (TGA), double outlet right ventricle (DORV) and tetralogy of Fallot (TOF). Treatment with NAC in drinking water in pregestational diabetic mice completely eliminated the incidence of AVSD, TGA, TOF and significantly diminished the incidence of ASD and VSD. Furthermore, pregestational diabetes increased ROS, impaired cell proliferation, and altered Gata4, Gata5 and Vegf-a expression in the fetal heart of diabetic offspring, which were all prevented by NAC treatment.

Conclusions: Treatment with NAC increases GSH levels, decreases ROS levels in the fetal heart and prevents the development of CHDs in the offspring of pregestational diabetes. Our study suggests that NAC may have therapeutic potential in the prevention of CHDs induced by pregestational diabetes.
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http://dx.doi.org/10.1186/1475-2840-13-46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942143PMC
February 2014

Nitric oxide synthase-3 promotes embryonic development of atrioventricular valves.

PLoS One 2013 29;8(10):e77611. Epub 2013 Oct 29.

Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.

Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3(-/-) mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3(-/-) compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3(-/-) mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1(+) cells in the AV cushion were decreased in NOS3(-/-) compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ), bone morphogenetic protein (BMP2) and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3(-/-) compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077611PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812218PMC
August 2014

Recombinant human annexin A5 inhibits proinflammatory response and improves cardiac function and survival in mice with endotoxemia.

Crit Care Med 2014 Jan;42(1):e32-41

1Department of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada. 2Centre for Critical Illness Research, Lawson Health Research Institute, London, ON, Canada. 3Department of Genetics, The Scripps Research Institute, La Jolla, CA. 4Department of Medicine, University of Western Ontario, London, ON, Canada.

Objectives: Annexin A5 is a 35-kDa protein with high affinity binding to negatively charged phospholipids. However, its effects on sepsis are not known. Our aim was to study the effects of annexin A5 on myocardial tumor necrosis factor-α expression, cardiac function, and animal survival in endotoxemia.

Design: Prospective experimental study.

Setting: University laboratory.

Subjects: Adult male C57BL/6 mice.

Interventions: Mice were challenged with lipopolysaccharide (4 or 20 mg/kg, i.p.) to induce endotoxemia with and without recombinant human annexin A5 treatment (5 or 10 μg/kg, i.v.). Cytokine expression and cardiac function were assessed, and animal survival was monitored.

Measurements And Main Results: Treatment with annexin A5 inhibited myocardial mitogen-activated protein kinase, and nuclear factor-κB activation in mice with endotoxemia. Furthermore, annexin A5-treated animals showed significant reductions in myocardial and plasma levels of tumor necrosis factor-α and interleukin-1β while cardiac function was significantly improved during endotoxemia. Additionally, 5-day animal survival was significantly improved by either an immediate or a 4-hour delayed annexin A5 treatment after lipopolysaccharide challenge. Importantly, annexin A5 dose-dependently inhibited lipopolysaccharide binding to a toll-like receptor-4/myeloid differentiation factor 2 fusion protein.

Conclusions: Annexin A5 treatment decreases cytokine expression and improves cardiac function and survival during endotoxemia. These effects of annexin A5 are mediated by its ability to inhibit lipopolysaccharide binding to toll-like receptor-4, leading to reductions in mitogen-activated protein kinase and Akt signaling. Our study suggests that annexin A5 may have therapeutic potential in the treatment of sepsis.
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http://dx.doi.org/10.1097/CCM.0b013e3182a63e01DOI Listing
January 2014

Cardiomyocyte-specific overexpression of human stem cell factor protects against myocardial ischemia and reperfusion injury.

Int J Cardiol 2013 Oct 13;168(4):3486-94. Epub 2013 May 13.

Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.

Background: Cardiomyocyte-specific overexpression of human membrane-associated stem cell factor (hSCF) improves cardiac function post-myocardial infarction. However, whether hSCF overexpression protects the heart from ischemia and reperfusion (I/R) injury is unknown. We aimed to investigate the effects of cardiomyocyte-specific overexpression of hSCF on cardiac injury after acute myocardial I/R and related cellular and molecular signaling mechanisms.

Methods And Results: Wild-type (WT) and hSCF/tetracycline transactivator (tTA) transgenic mice (hSCF/tTA) were subjected to myocardial ischemia for 45 min followed by 3 h of reperfusion. Infarct size and myocardial apoptosis were decreased in hSCF/tTA compared to WT mice (P<0.05). Furthermore, these cardioprotective effects in the hSCF/tTA mice were abrogated by doxycycline, which turned off hSCF overexpression, and by a PI3 kinase inhibitor LY294002. Myocardial expression of insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF), which are upstream activators of Akt signaling, was significantly increased in hSCF/tTA compared to WT mice after I/R (P<0.05), and was associated with higher number of c-kit(+) cardiac stem cells (CSCs) (P<0.05). Inhibition of c-kit signaling by ACK2 treatment abolished these protective effects in hSCF/tTA mice.

Conclusions: Cardiomyocyte-specific overexpression of hSCF protects the heart from I/R injury. The cardioprotective effects of hSCF overexpression are mediated by increased c-kit(+) CSCs, enhanced growth factor expression and activation of Akt signaling pathway.
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http://dx.doi.org/10.1016/j.ijcard.2013.04.165DOI Listing
October 2013

Nitric oxide synthase-3 deficiency results in hypoplastic coronary arteries and postnatal myocardial infarction.

Eur Heart J 2014 Apr 9;35(14):920-31. Epub 2012 Oct 9.

Department of Physiology and Pharmacology, Western University, London, Ontario, Canada N6A 5C1.

Aims: Hypoplastic coronary artery disease is a rare congenital abnormality that is associated with sudden cardiac death. However, molecular mechanisms responsible for this disease are not clear. The aim of the present study was to assess the role of nitric oxide synthase-3 (NOS3) in the pathogenesis of hypoplastic coronary arteries.

Methods And Results: Wild-type (WT), NOS3(-/-), and a novel cardiac-specific NOS3 overexpression mouse model were employed. Deficiency in NOS3 resulted in coronary artery hypoplasia in foetal mice and spontaneous myocardial infarction in postnatal hearts. Coronary artery diameters, vessel density, and volume were significantly decreased in NOS3(-/-) mice at postnatal day 0. In addition, NOS3(-/-) mice showed a significant increase in the ventricular wall thickness, myocardial volume, and cardiomyocyte cell size compared with WT mice. Lack of NOS3 also down-regulated the expression of Gata4, Wilms tumour-1, vascular endothelial growth factor, basic fibroblast growth factor and erythropoietin, and inhibited migration of epicardial cells. These abnormalities and hypoplastic coronary arteries in the NOS3(-/-) mice were completely rescued by the cardiac-specific overexpression of NOS3.

Conclusion: Nitric oxide synthase-3 is required for coronary artery development and deficiency in NOS3 leads to hypoplastic coronary arteries.
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http://dx.doi.org/10.1093/eurheartj/ehs306DOI Listing
April 2014

Inhibition of Na/K-ATPase promotes myocardial tumor necrosis factor-alpha protein expression and cardiac dysfunction via calcium/mTOR signaling in endotoxemia.

Basic Res Cardiol 2012 Mar 21;107(2):254. Epub 2012 Feb 21.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, N6A 5C1, Canada.

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that causes cardiac dysfunction during sepsis. Na/K-ATPase regulates intracellular Ca(2+), which activates mammalian target of rapamycin (mTOR), a regulator of protein synthesis. The aim of this study was to investigate the role of Na/K-ATPase/mTOR signaling in myocardial TNF-α expression during endotoxemia. Results showed that treatment with LPS decreased Na/K-ATPase activity in the myocardium in vivo and in cultured neonatal cardiomyocytes. Inhibition of Na/K-ATPase by ouabain enhanced LPS-induced myocardial TNF-α protein production, but had no effect on TNF-α mRNA expression. More importantly, ouabain further decreased in vivo cardiac function in endotoxemic mice, which was blocked by etanercept, a TNF-α antagonist. LPS-induced reduction in Na/K-ATPase activity was prevented by inhibition of PI3K, Rac1 and NADPH oxidase using LY294002, a dominant-negative Rac1 adenovirus (Ad-Rac1N17) and apocynin, respectively. To assess the role of Rac1 in Ca(2+) handling, Ca(2+) transients in adult cardiomyocytes from cardiomyocyte-specific Rac1 knockout (Rac1(CKO)) and wild-type (WT) mice were determined. LPS increased intracellular Ca(2+) in WT but not in Rac1(CKO) cardiomyocytes. Furthermore, LPS rapidly increased mTOR phosphorylation in cardiomyocytes, which was blocked by Rac1N17 and an inhibitor of calmodulin-dependent protein kinases (CaMKs) KN93, but enhanced by ouabain. Rapamycin, an inhibitor of mTOR suppressed TNF-α protein levels without any significant effect on its mRNA expression or global protein synthesis. In conclusion, myocardial Na/K-ATPase activity is inhibited during endotoxemia via PI3K/Rac1/NADPH oxidase activation. Inhibition of Na/K-ATPase activates Ca(2+)/CaMK/mTOR signaling, which promotes myocardial TNF-α protein production and cardiac dysfunction during endotoxemia.
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http://dx.doi.org/10.1007/s00395-012-0254-8DOI Listing
March 2012

Mitogen-activated protein kinase phosphatase-1 inhibits myocardial TNF-α expression and improves cardiac function during endotoxemia.

Cardiovasc Res 2012 Mar 23;93(3):471-9. Epub 2011 Dec 23.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

Aims: Myocardial tumour necrosis factor-α (TNF-α) expression induces cardiac dysfunction in endotoxemia. The aim of this study was to investigate the role of mitogen-activated protein kinase phosphatase-1 (MKP1) pathway in myocardial TNF-α expression and cardiac function during endotoxemia.

Methods And Results: Lipopolysaccharide (LPS) increased MKP1 expression in the myocardium in vivo and in cultured neonatal cardiomyocytes in vitro. LPS-induced extracellular signal-regulated kinase (ERK) 1/2 and p38 phosphorylation in the myocardium was prolonged in MKP1(-/-) mice. Myocardial TNF-α mRNA and protein levels were enhanced in MKP1(-/-) compared with wild-type (WT) mice in endotoxemia, leading to a further decrease in cardiac function. To study if Rac1/p21-activated kinase 1 (PAK1) signalling regulates MKP1 expression, cardiomyocytes were treated with LPS. Inhibition of Rac1 and PAK1 by a dominant negative Rac1 adenovirus (Ad-Rac1N17) and PAK1 siRNA, respectively, blocked LPS-induced MKP1 expression in cardiomyocytes. PAK1 siRNA also decreased p38 and c-Jun N-terminal kinase (JNK) activation, and TNF-α expression induced by LPS. Furthermore, deficiency in either Rac1 or JNK1 decreased myocardial MKP1 expression in endotoxemic mice.

Conclusion: LPS activates the Rac1/PAK1 pathway, which increases myocardial MKP1 expression via JNK1. MKP1 attenuates ERK1/2 and p38 activation, inhibits myocardial TNF-α expression, and improves cardiac function in endotoxemia. Thus, MKP1 represents an important negative feedback mechanism limiting pro-inflammatory response in the heart during sepsis.
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http://dx.doi.org/10.1093/cvr/cvr346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282576PMC
March 2012

Transfusion of fresh but not old stored blood reduces infarct size and improves cardiac function after acute myocardial infarction in anemic rats*.

Crit Care Med 2012 Mar;40(3):740-6

Centre for Critical Illness, Lawson Research Health Research Institute, University of Western Ontario, London, Ontario, Canada.

Objectives: We recently demonstrated that transfusion of fresh blood to 100 g/L hemoglobin in anemic animals offers cardioprotection after acute myocardial infarction. The objective of this study was to compare the cardioprotective effects of fresh vs. stored blood when transfused in anemic rats after acute myocardial infarction.

Study Design: Randomized animal study.

Setting: University laboratory.

Subjects: Male Sprague-Dawley rats weighing 200-300 g.

Intervention: Myocardial infarction was induced by coronary artery ligation in 49 male Sprague-Dawley rats weighing 200-300 g, 38 of which were anemic (80-90 g/L) and 11 with normal hemoglobin levels. Anemic animals were randomized to receive fresh blood (within 4 hrs), stored blood (7 days), or no transfusion immediately after myocardial infarction.

Measurements And Main Results: At 24 hrs after myocardial infarction, cardiac function, infarct size, and apoptosis were determined. Erythrocyte ATP, 2,3-DPG, hemoglobin, and free hemoglobin levels in the supernatant were determined. Transfusion with fresh but not stored blood significantly decreased infarct size and myocardial apoptosis in anemic rats when compared to anemic animals not undergoing transfusion. Cardiac function and survival were significantly improved in the anemic animals undergoing fresh blood transfusion compared to control anemic animals. Analysis of stored red blood cells showed reductions of intracellular ATP and 2,3-DPG levels and free hemoglobin was increased in the supernatant.

Conclusions: The prolonged storage of blood negates the beneficial effects of fresh blood transfusion, which include reductions in infarct size and myocardial apoptosis, and improvements in cardiac function and short-term survival after acute myocardial infarction in this animal model.
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http://dx.doi.org/10.1097/CCM.0b013e3182376e84DOI Listing
March 2012

North American ginseng protects the heart from ischemia and reperfusion injury via upregulation of endothelial nitric oxide synthase.

Pharmacol Res 2011 Sep 19;64(3):195-202. Epub 2011 May 19.

Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, China.

Emerging evidence suggests ginseng has therapeutic potential in cardiovascular disease. The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the cardioprotective effects of ginseng during myocardial ischemia and reperfusion (I/R). Treatment with ginseng extract significantly increased Akt phosphorylation and eNOS protein levels in cultured neonatal cardiomyocytes. Upregulation of eNOS was blocked by LY294002, a PI3-kinase inhibitor, suggesting a PI3-kinase/Akt-dependent mechanism. To simulate I/R, cultured neonatal cardiomyocytes from eNOS(-/-) and wild-type (WT) mice were subjected to anoxia and reoxygenation (A/R). Ginseng treatment inhibited A/R-induced apoptosis in WT, but not in either eNOS(-/-) cardiomyocytes or WT cardiomyocytes treated with LY294002. To further study the cardioprotective effects of ginseng in vivo, WT and eNOS(-/-) mice were pretreated with ginseng extract (50mg/kg/day, oral gavage) for 7 days before they were subjected to myocardial I/R. Treatment with ginseng significantly increased Akt phosphorylation and eNOS protein levels in the myocardium. Furthermore, ginseng-induced myocardial eNOS expression was inhibited by LY294002. Strikingly, ginseng treatment significantly decreased infarct size and myocardial apoptosis following I/R in WT mice, but not in either eNOS(-/-) mice or WT mice treated with LY294002. We conclude that ginseng treatment protects the heart from I/R injury via upregulation of eNOS expression. Our study suggests that ginseng may serve as a potential therapeutic agent to limit myocardial I/R injury.
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http://dx.doi.org/10.1016/j.phrs.2011.05.006DOI Listing
September 2011

Deficiency in TIMP-3 increases cardiac rupture and mortality post-myocardial infarction via EGFR signaling: beneficial effects of cetuximab.

Basic Res Cardiol 2011 May 18;106(3):459-71. Epub 2011 Jan 18.

Department of Physiology and Pharmacology, University of Western Ontario, London, Canada.

Cardiac rupture is a fatal complication of myocardial infarction (MI); however, its underlying molecular mechanisms are not fully understood. This study investigated the role of tissue inhibitor of metalloproteinase-3 (TIMP-3)/matrix metalloproteinase (MMP)/epidermal growth factor (EGF)/transforming growth factor (TGF)-β1 pathway in infarct healing and effects of cetuximab on cardiac rupture after MI. Induction of MI was achieved by left coronary artery ligation in wild-type (WT) and TIMP-3(-/-) mice. TIMP-3 deficiency resulted in a fourfold increase in cardiac rupture and 50% decrease in survival after MI. Hydroxyproline content, collagen synthesis and myofibroblast cell number in the infarct region, and the force required to induce rupture of the infarct scar were significantly decreased, while MMP activity was increased in TIMP-3(-/-) mice. EGF proteins were increased by threefold in TIMP-3(-/-) mice following MI, while TGF-β1 mRNA levels were decreased by 68%. Cell proliferation of cultured adult cardiac myofibroblasts was significantly decreased in TIMP-3(-/-) compared to WT myofibroblasts. EGF treatment significantly decreased collagen synthesis and TGF-β1 expression. Conversely, TGF-β1 treatment increased collagen synthesis in cardiac myofibroblasts. Treatment with cetuximab significantly decreased the incidence of cardiac rupture and improved survival post-MI in TIMP-3(-/-) mice. We conclude that deficiency in TIMP-3 increases cardiac rupture post-MI via EGF/epidermal growth factor receptor (EGFR) signaling which downregulates TGF-β1 expression and collagen synthesis. Inhibition of EGFR by cetuximab protects against cardiac rupture and improves survival post-MI.
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http://dx.doi.org/10.1007/s00395-010-0147-7DOI Listing
May 2011

Erythropoietin is equally effective as fresh-blood transfusion at reducing infarct size in anemic rats.

Crit Care Med 2010 Nov;38(11):2215-21

Centre for Critical Illness, Research Lawson Health Research Institute, Canadian Blood Services, Ontario, Canada.

Objective: We recently demonstrated that transfusion of anemic animals up to 100 g/L hemoglobin with fresh blood protects the heart from ischemic injuries following myocardial infarction. Erythropoietin has cardioprotective effects independent of its erythropoietic activity. The objective of this study was to compare the cardioprotective effects of erythropoietin treatment to fresh-blood transfusion in anemic rats after acute myocardial infarction.

Design: Randomized animal study.

Setting: University laboratory.

Subjects: Male Sprague-Dawley rats weighing 200-300 g.

Intervention: Myocardial infarction was induced by coronary artery ligation in 76 rats, 55 of which were anemic (80-90 g/L) and 21 of which had normal hemoglobin levels. Animals were randomized to erythropoietin (2000 units/kg), fresh-blood transfusion to 100 g/L hemoglobin, or saline-treatment groups immediately following myocardial infarction.

Measurements And Main Results: At 24 hrs after myocardial infarction, cardiac function and infarct size were determined. Myocardial apoptosis was determined by caspase-3 activity and terminal deoxynucleotidyl transferase d-UTP nick end labeling (TUNEL) assay. Infarct size was significantly decreased in anemic rats treated with erythropoietin or blood transfusion compared to those in the saline-treatment group. Cardiac function, as measured by maximal positive and minimal negative first derivatives of left ventricular pressure, was better preserved in the normal hemoglobin groups and the erythropoietin- or transfusion-treated anemic animals compared to saline-treated anemic animals. Myocardial caspase-3 activity and TUNEL-positive nuclei were significantly increased in anemic rats but were decreased by erythropoietin treatment or red blood cell transfusion.

Conclusions: Erythropoietin treatment is equally effective as fresh-blood transfusion in anemic rats after acute myocardial infarction at reducing infarct size, myocardial apoptosis, and improving cardiac function.
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http://dx.doi.org/10.1097/CCM.0b013e3181f17d6eDOI Listing
November 2010

NOX2 deficiency protects against streptozotocin-induced beta-cell destruction and development of diabetes in mice.

Diabetes 2010 Oct 13;59(10):2603-11. Epub 2010 Jul 13.

Department of Medicine, University of Western Ontario, Lawson Health Research Institute, London, Ontario, Canada.

Objective: The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice.

Research Design And Methods: Five groups of mice--wild-type (WT), NOX2(-/-), WT treated with apocynin, and WT adoptively transferred with NOX2(-/-) or WT splenocytes--were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2(-/-) pancreatic islets were treated with cytokines for 48 h.

Results: Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2(-/-) mice and in WT mice adoptively transferred with NOX2(-/-) splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2(-/-) mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2(-/-) compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2(-/-) compared with WT splenocytes.

Conclusions: NOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis.
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http://dx.doi.org/10.2337/db09-1562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279537PMC
October 2010

Rac1 activation induces tumour necrosis factor-α expression and cardiac dysfunction in endotoxemia.

J Cell Mol Med 2011 May 26;15(5):1109-21. Epub 2010 May 26.

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

Induction of tumour necrosis factor-α (TNF-α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF-α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide-3 kinase (PI3K) in lipopolysaccharide (LPS)-induced Rac1 activation. Our results showed that LPS-induced Rac1 activation and TNF-α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant-negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF-α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS-induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild-type and cardiomyocyte-specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF-α expression and improved cardiac function during endotoxemia. We conclude that PI3K-mediated Rac1 activation is required for induction of TNF-α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF-α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.
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http://dx.doi.org/10.1111/j.1582-4934.2010.01095.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822624PMC
May 2011

Comparison of initial cell retention and clearance kinetics after subendocardial or subepicardial injections of endothelial progenitor cells in a canine myocardial infarction model.

J Nucl Med 2010 Mar 11;51(3):413-7. Epub 2010 Feb 11.

Department of Medical Biophysics, University of Western Ontario, Ontario, Canada.

Unlabelled: Neither intravenous nor intracoronary routes provide targeted stem cell delivery to recently infarcted myocardium in sufficient quantities. Direct routes appear preferable. However, most prior studies have used epicardial injections, which are not practical for routine clinical use. The objective of this study was to compare cell retention and clearance kinetics between a subepicardial and a subendocardial technique.

Methods: We evaluated 7 dogs with each technique, using (111)In-tropolone-labeled endothelial progenitor cells and serial SPECT/CT for 15 d after injection.

Results: In vivo indium imaging demonstrated comparable degrees of retention: 57% +/- 15% for the subepicardial injections and 54% +/- 26% for the subendocardial injections. Clearance half-lives were also similar at 69 +/- 26 and 60 +/- 21 h, respectively.

Conclusion: This study demonstrates that subendocardial injections, clinically more practical, show clearance kinetics comparable to those of subepicardial injections and will facilitate the ultimate clinical use of this treatment modality.
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http://dx.doi.org/10.2967/jnumed.109.069732DOI Listing
March 2010
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