Maternal nicotine exposure induces congenital heart defects in the offspring of mice.

Maternal cigarette smoking is a risk factor for congenital heart defects (CHDs). Nicotine replacement therapies are often offered to pregnant women following failed attempts of smoking cessation. However, the impact of nicotine on embryonic heart development is not well understood. In the present study, the effects of maternal nicotine exposure (MNE) during pregnancy on foetal heart morphogenesis were studied. Adult female mice were treated with nicotine using subcutaneous osmotic pumps at 0.75 or 1.5 mg/kg/day and subsequently bred with male mice. Our results show that MNE dose-dependently increased CHDs in foetal mice. CHDs included atrial and ventricular septal defects, double outlet right ventricle, unguarded tricuspid orifice, hypoplastic left ventricle, thickened aortic and pulmonary valves, and ventricular hypertrophy. MNE also significantly reduced coronary artery size and vessel abundance in foetal hearts. Moreover, MNE resulted in higher levels of oxidative stress and altered the expression of key cardiogenic regulators in the developing heart. Nicotine exposure reduced epicardial-to-mesenchymal transition in foetal hearts. In conclusion, MNE induces CHDs and coronary artery malformation in mice. These findings provide insight into the adverse outcomes of foetuses by MNE during pregnancy.

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

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, ß-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.

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

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.

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

Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway facilitating extracellular Ca2+ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca2+ stores. Stromal interaction molecule 1 (STIM1) is the Ca2+ sensor that activates SOCE following ER/SR Ca2+ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2+ concentrations. Low ER Ca2+ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2+ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2+ 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 S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56S-nitrosylation 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 S-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.

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

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.

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.

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 Gq- and Gs- 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 (RGS2eb) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed in neonatal rat cardiomyocytes, RGS2eb attenuates both protein synthesis and hypertrophy induced by Gq- and Gs- activating agents. In the current study, we investigated the potential cardioprotective role of RGS2eb by determining whether RGS2eb transgenic (RGS2eb TG) mice with cardiomyocyte specific overexpression of RGS2eb 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 RGS2eb TG mice compared to WT controls following four weeks of TAC. Expression of the hypertrophic markers atrial natriuretic peptide (ANP) and ß-myosin heavy chain (MHC-ß) was also reduced in RGS2eb TG compared to WT TAC animals. Furthermore, cardiac function in RGS2eb 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.

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

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.

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

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.

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

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.

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

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-1ß 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.

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

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.

Novel circRNA-miRNA-mRNA networks regulated by maternal exercise in fetal hearts of pregestational diabetes.

BACKGROUND: Maternal exercise lowers the incidence of congenital heart defects (CHDs) induced by pregestational diabetes. However, the molecular mechanisms underlying the beneficial effects of maternal exercise remain unclear. The present study aimed to identify circular RNA (circRNA), microRNA (miRNA) and mRNA networks that are regulated by maternal exercise in fetal hearts of pregestational diabetes. METHODS: Pregestational diabetes was induced in adult C57BL/6 female mice by streptozotocin. The expression profiles of circRNAs, miRNAs and mRNAs in E10.5 fetal hearts of offspring of control and diabetic mothers with or without exercise were analyzed using next generation sequencing. circRNA-miRNA-mRNA networks in fetal hearts were mapped and key candidate transcripts were verified by qPCR analysis. RESULTS: Pregestational diabetes dysregulated the expression of 206 circRNAs, 66 miRNAs and 391 mRNAs in fetal hearts. Maternal exercise differentially regulated 188 circRNAs, 57 miRNAs and 506 mRNAs in fetal hearts of offspring of pregestational diabetes. A total of 5 circRNAs, 12 miRNAs, and 28 mRNAs were incorporated into a final maternal exercise-associated regulatory network in fetal hearts of offspring of maternal diabetes. Notably, maternal exercise normalized the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs in fetal hearts of pregestational diabetes. CONCLUSION: Maternal exercise reverses the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs, and partially normalizes circRNA, miRNA, and mRNA expression profiles in fetal hearts of pregestational diabetes. These findings shed new light on the potential mechanisms of the beneficial effects of maternal exercise on the developing heart in diabetic pregnancies.

Annexin A5 Inhibits Endothelial Inflammation Induced by Lipopolysaccharide-Activated Platelets and Microvesicles via Phosphatidylserine Binding.

Sepsis is caused by a dysregulated immune response to infection and is a leading cause of mortality globally. To date, no specific therapeutics are available to treat the underlying septic response. We and others have shown that recombinant human annexin A5 (Anx5) treatment inhibits pro-inflammatory cytokine production and improves survival in rodent sepsis models. During sepsis, activated platelets release microvesicles (MVs) with externalization of phosphatidylserine to which Anx5 binds with high affinity. We hypothesized that recombinant human Anx5 blocks the pro-inflammatory response induced by activated platelets and MVs in vascular endothelial cells under septic conditions via phosphatidylserine binding. Our data show that treatment with wildtype Anx5 reduced the expression of inflammatory cytokines and adhesion molecules induced by lipopolysaccharide (LPS)-activated platelets or MVs in endothelial cells (p < 0.01), which was not observed with Anx5 mutant deficient in phosphatidylserine binding. In addition, wildtype Anx5 treatment, but not Anx5 mutant, improved trans-endothelial electrical resistance (p < 0.05) and reduced monocyte (p < 0.001) and platelet (p < 0.001) adhesion to vascular endothelial cells in septic conditions. In conclusion, recombinant human Anx5 inhibits endothelial inflammation induced by activated platelets and MVs in septic conditions via phosphatidylserine binding, which may contribute to its anti-inflammatory effects in the treatment of sepsis.

Pharmacokinetics of recombinant human annexin A5 (SY-005) in patients with severe COVID-19.

Objective: Annexin A5 is a phosphatidylserine binding protein with anti-inflammatory, anticoagulant and anti-apoptotic properties. Preclinical studies have shown that annexin A5 inhibits pro-inflammatory responses and improves organ function and survival in rodent models of sepsis. This clinical trial aimed to evaluate the pharmacokinetic (PK) properties of the recombinant human annexin A5 (SY-005) in severe COVID-19. Methods: This was a pilot randomized, double-blind, placebo-controlled trial. Severe COVID-19 patients were randomly assigned to receive intravenous 50 µg/kg (low dose, n = 3), 100 µg/kg (high dose, n = 5) of SY-005 or placebo (n = 5) every 12 h for 7 days. Plasma SY-005 levels were assessed using enzyme-linked immunosorbent assay (ELISA) and the PK parameters were determined using non-compartmental analysis. Results: All patients treated with SY-005 had a normal baseline estimated glomerular filtration rate (eGFR, 104-125 mL/min/1.73 m2). Both low and high doses of SY-005 were cleared within 6 h after intravenous administration. Plasma maximum concentrations (Cmax), half-life, clearance and volume distribution of low and high doses of SY-005 were 402.4 and 848.9 ng/mL, 0.92 and 0.96 h, 7.52 and 15.19 L/h, and 9.98 and 20.79 L, respectively. Daily pre-dose circulating annexin A5 levels were not significantly different when SY-005 was administered at the low or the high dose 12-h intervals. There was no significant effect on activated partial thromboplastin time (aPTT) or INR (international normalized ratio of prothrombin time) during 7 days of SY-005 treatment. Conclusion: SY-005 doses of 50 and 100 µg/kg were detectable and subsequently cleared from the plasma in severe COVID-19 patients with normal baseline renal function. There was no significant plasma SY-005 accumulation 6 h after drug administration and coagulation was not altered during 7 days of treatment. Clinical trials Registration: This study was registered with ClinicalTrials.gov (NCT04748757, first posted on 10 February 2021).

Annexin A5 in Patients With Severe COVID-19 Disease: A Single-Center, Randomized, Double-Blind, Placebo-Controlled Feasibility Trial.

OBJECTIVES: To evaluate the study design and feasibility of drug administration and safety in a randomized clinical trial of recombinant human annexin A5 (SY-005), a constitutively expressed protein with anti-inflammatory, antiapoptotic, and anticoagulant properties, in patients with severe coronavirus disease 2019 (COVID-19). DESIGN: Double-blind, randomized clinical trial. SETTING: Two ICUs at an academic medical center. PATIENTS/SUBJECTS: Adults admitted to the ICU with a confirmed diagnosis of COVID-19 and requiring ventilatory or vasopressor support. INTERVENTIONS: SY-005, a recombinant human annexin A5, at 50 or 100 µg/kg IV every 12 hours for 7 days. MEASUREMENTS AND MAIN RESULTS: We enrolled 18 of the 55 eligible patients (33%) between April 21, 2021, and February 3, 2022. We administered 82% (196/238) of the anticipated doses of study medication and 86% (169/196) were given within 1 hour of the scheduled time. There were no drug-related serious adverse events. We captured 100% of the data that would be required for measuring clinical outcomes in a phase 2 or 3 trial. LIMITATIONS: The small sample size was a result of decreasing admissions of patients with COVID-19, which triggered a stopping rule for the trial. CONCLUSIONS: Although enrollment was low, administration of SY-005 to critically ill patients with COVID-19 every 12 hours for up to 7 days was feasible and safe. Further clinical trials of annexin A5 for the treatment of COVID-19 are warranted. Given reduction of severe COVID-19 disease, future studies should explore the safety and effectiveness of SY-005 use in non-COVID-related sepsis.

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

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.

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

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.

Maternal Delta-9-Tetrahydrocannabinol Exposure Induces Abnormalities of the Developing Heart in Mice.

Introduction: Cannabis is increasingly being consumed by pregnant women for recreational purposes as well as for its antiemetic and anxiolytic effects despite limited studies on its safety during pregnancy. Importantly, phytocannabinoids found in cannabis can pass through the placenta and enter the fetal circulation. Recent reports suggest gestational cannabis use is associated with negative fetal outcomes, including fetal growth restriction and perinatal intensive care, however, the effects of delta-9-tetrahydrocannabinol (THC) on fetal heart development remains to be elucidated. Materials and Methods: We aimed to determine the outcomes of maternal THC exposure on fetal heart development in mice by administering 0, 5, or 10 mg/kg/day of THC orally to C57BL/6 dams starting at embryonic day (E)3.5. Offspring were collected at E12.5 for molecular analysis, at E17.5 to analyze cardiac morphology or at postnatal day (PND)21 to assess heart function. Results: Maternal THC exposure in E17.5 fetuses resulted in an array of cardiac abnormalities with an incidence of 44% and 55% in the 5 and 10 mg/kg treatment groups, respectively. Maternal THC exposure in offspring resulted in ventricular septal defect, higher semilunar valve volume relative to orifice ratio, and higher myocardial wall thickness. Notably, cell proliferation within the ventricular myocardium was increased, and expression of multiple cardiac transcription factors was downregulated in THC-exposed E12.5 fetuses. Furthermore, heart function was compromised with lower left ventricular ejection fraction, fractional shortening, and cardiac output in PND21 pups exposed to THC compared to controls. Discussion: The results show that maternal THC exposure during gestation induces myocardial hyperplasia and semilunar valve thickening in the fetal heart and postnatal cardiac dysfunction. Our study suggests that maternal cannabis consumption may induce abnormalities in the developing heart and cardiac dysfunction in postnatal life.

Inhibition of intraflagellar transport protein-88 promotes epithelial-to-mesenchymal transition and reduces cardiac remodeling post-myocardial infarction.

The epicardium is a potential source of cardiac progenitors to support reparative angiogenesis after myocardial infarction (MI) through epithelial-to-mesenchymal transition (EMT). Primary cilia are recognized as hubs of cellular signaling, and their presence can alter downstream pathways to modulate EMT. The present study aimed to examine the effects of inhibiting intraflagellar transport protein-88 (Ift88), a protein vital to ciliary assembly, on epicardial EMT and cardiac remodeling post-MI. Epicardium derived cells (EPDCs) were cultured from E13.5 heart explants and treated with adenoviral vector encoding short-hairpin RNA against the mouse Ift88 (Ad-shIft88) to disassemble the primary cilium. Effects of Ad-shIft88 on epicardial EMT and cardiac remodeling were examined in mice post-MI. Our results show that Ad-shIft88 enhanced EMT of cultured EPDCs. In adult mice, intra-myocardial administration of Ad-shIft88 increased the number of Wilms tumor 1 (Wt1) positive cells in the epicardium and myocardium, promoted expression of genes associated with epicardial EMT, and enhanced capillary and arteriolar densities post-MI. Additionally, intra-myocardial Ad-shIft88 treatment attenuated cardiac hypertrophy and improved myocardial function three weeks post-MI. In conclusion, knockdown of Ift88 improves epicardial EMT, neovascularization and cardiac remodeling in the ischemic heart. Our study highlights the primary cilium as a potential therapeutic target post-MI.

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

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.