IntroductionKawasaki disease (KD) is a diffuse vasculitis in children. Response to high dose intravenous gamma globulin (IVIG), the primary treatment, varies according to genetic background. We sought to identify genetic loci, which associate with treatment response using whole genome sequencing (WGS).MethodWe performed WGS in 472 KD patients with 305 IVIG responders and 167 non-responders defined by AHA clinical criteria. We conducted logistic regression models to test additive genetic effect in the entire cohort and in four subgroups defined by ancestry information markers (Whites, African Americans, Asians, and Hispanics). We performed functional mapping and annotation using FUMA to examine genetic variants that are potentially involved IVIG non-response. Further, we conducted SNP-set [Sequence] Kernel Association Test (SKAT) for all rare and common variants.ResultsOf the 43,288,336 SNPs (23,660,970 in intergenic regions, 16,764,594 in introns and 556,814 in the exons) identified, the top ten hits associated with IVIG non-response were in FANK1, MAP2K3:KCNJ12, CA10, FRG1DP, CWH43 regions. When analyzed separately in ancestry-based racial subgroups, SNPs in several novel genes were associated. A total of 23 possible causal genes were pinpointed by positional and chromatin mapping. SKAT analysis demonstrated association in the entire MANIA2, EDN1, SFMBT2, and PPP2R5E genes and segments of CSMD2, LINC01317, HIVEPI, HSP90AB1, and TTLL11 genesConclusionsThis WGS study identified multiple predominantly novel understudied genes associated with IVIG response. These data can serve to inform regarding pathogenesis of KD, as well as lay ground work for developing treatment response predictors.

Aerosolized lung surfactant therapy during nasal continuous positive airway pressure (CPAP) support avoids intubation but is highly complex, with reported poor nebulizer efficiency and low pulmonary deposition. The study objective was to evaluate particle size, operational compatibility, and drug delivery efficiency with various nasal CPAP interfaces and gas humidity levels of a synthetic dry powder (DP) surfactant aerosol delivered by a low-flow aerosol chamber (LFAC) inhaler combined with bubble nasal CPAP (bCPAP). A particle impactor characterized DP surfactant aerosol particle size. Lung pressures and volumes were measured in a preterm infant nasal airway and lung model using LFAC flow injection into the bCPAP system with different nasal prongs. The LFAC was combined with bCPAP and a non-heated passover humidifier. DP surfactant mass deposition within the nasal airway and lung was quantified for different interfaces. Finally, surfactant aerosol therapy was investigated using select interfaces and bCPAP gas humidification by active heating. Surfactant aerosol particle size was 3.68 µm. Lung pressures and volumes were within an acceptable range for lung protection with LFAC actuation and bCPAP. Aerosol delivery of DP surfactant resulted in variable nasal airway (0–20%) and lung (0–40%) deposition. DP lung surfactant aerosols agglomerated in the prongs and nasal airways with significant reductions in lung delivery during active humidification of bCPAP gas. Our findings show high-efficiency delivery of small, synthetic DP surfactant particles without increasing the potential risk for lung injury during concurrent aerosol delivery and bCPAP with passive humidification. Specialized prongs adapted to minimize extrapulmonary aerosol losses and nasal deposition showed the greatest lung deposition. The use of heated, humidified bCPAP gases compromised drug delivery and safety. Safety and efficacy of DP aerosol delivery in preterm infants supported with bCPAP requires more research.

Protein posttranslational modifications (PTMs) by O-GlcNAc globally rise during pressure-overload hypertrophy (POH). However, a major knowledge gap exists on the specific proteins undergoing changes in O-GlcNAc levels during POH primarily because this PTM is low abundance and easily lost during standard mass spectrometry (MS) conditions used for protein identification. Methodologies have emerged to enrich samples for O-GlcNAcylated proteins prior to MS analysis. Accordingly, our goal was to identify the specific proteins undergoing changes in O-GlcNAc levels during POH. We used C57/Bl6 mice subjected to Sham or transverse aortic constriction (TAC) to create POH. From the hearts, we labelled the O-GlcNAc moiety with tetramethylrhodamine azide (TAMRA) before sample enrichment by TAMRA immunoprecipitation (IP). We used LC-MS/MS to identify and quantify the captured putative O-GlcNAcylated proteins. We identified a total of 700 putative O-GlcNAcylated proteins in Sham and POH. Two hundred thirty-three of these proteins had significantly increased enrichment in POH over Sham suggesting higher O-GlcNAc levels whereas no proteins were significantly decreased by POH. We examined two MS identified metabolic enzymes, CPT1B and the PDH complex, to validate by immunoprecipitation. We corroborated increased O-GlcNAc levels during POH for CPT1B and the PDH complex. Enzyme activity assays suggests higher O-GlcNAcylation increases CPT1 activity and decreases PDH activity during POH. In summary, we generated the first comprehensive list of proteins with putative changes in O-GlcNAc levels during POH. Our results demonstrate the large number of potential proteins and cellular processes affected by O-GlcNAc and serve as a guide for testing specific O-GlcNAc-regulated mechanisms during POH.

Introduction: Kawasaki Disease (KD) leads as a cause of acquired cardiovascular disease among children in developed countries. Around~20% of patients are refractory to high-dose intravenous immunoglobulin (IVIG), the standard treatment, and are at high risk of developing coronary artery aneurysms. Hypothesis: Whole genome sequencing (WGS) will identify specific predictors of non-response and inform the mechanism of IVIG action. Methods: KD patients were diagnosed by the American Heart Association and the American Academy of Pediatrics (AHA/AAP) criteria and treated by AHA guidelines. IVIG refractoriness was defined by persistent or recurrent fever (>38C)>36 hours after completing infusion. We performed a case-control study with WGS on DNA from saliva or blood from 107 responders versus 103 non-responders. After processing informatics using the pipeline of Genome Analysis Toolkit (GATK), additive genetic models were assessed for all common variants using logistic regression. We adjusted for age, gender, and race based on principal components. We also conducted SNP-set (Sequence) Kernel Association Test (SKAT) with common and rare variants in the gene regions with the most statistically significant SNP results. Results: Average sequencing depth was 30x and with a total of 34,548,910 variants. While SNPs in genes previously indicated in the IVIG pathway were confirmed, multiple novel SNPs in various gene regions were also statistically significant (often p< 10-6; Figure-Manhattan plot). SKAT analysis suggested joint effects of multiple SNPs in individual gene regions. Conclusions: We confirmed SNPs in genes from previous reports and identified multiple novel ones, indicating plausible previous unsuspected IVIG mechanisms pathways. Potentially, functional disruption of these genes could lead to non-response. Further, a panel of these SNPs can be a prognostic tool for identifying at-risk KD patients that require therapy intensification.

Thyroid hormones (THs) play a critical role in the metabolic phenotype of the heart; and most of the effects involve transcriptional regulation via thyroid hormone receptors (TRs). TRs ability to form combinatorial complexes with an array of partners accounts for TRs physiological flexibility in modulating gene expression. To identify proteins that associate with TRβ1 in the heart we performed a pull-down assay on cardiac tissue using GST-TRβ1 as bait and identified the bound proteins by LC MS/MS. ACAA2, a mitochondrial thiolase enzyme, was identified as a novel interacting protein. We confirmed ACAA2 localized to the nucleus and using a luciferase reporter assay showed ACAA2 acted as a TH-dependent coactivator for TRβ1. ACAA2 showed an ability to bind to TR recognition sequences but did not alter TRβ1 DNA binding ability. Thus, ACAA2 as a novel TRβ1 associating protein opens a new paradigm to understanding how TH/TRs may be manipulated by energetic pathway molecules.

Protein posttranslational modifications (PTMs) by O-linked beta-N-acetylglucosamine (O-GlcNAc) rise during pressure-overload hypertrophy (POH) to affect hypertrophic growth. The hexosamine biosynthesis pathway (HBP) branches from glycolysis to make the moiety for O-GlcNAcylation. It is speculated that greater glucose utilization during POH augments HBP flux to increase O-GlcNAc levels; however, recent results suggest glucose availability does not primarily regulate cardiac O-GlcNAc levels. We hypothesize that induction of key enzymes augment protein O-GlcNAc levels primarily during active myocardial hypertrophic growth and remodeling with early pressure overload. We further speculate that downregulation of protein O-GlcNAcylation inhibits ongoing hypertrophic growth during prolonged pressure overload with established hypertrophy. We used transverse aortic constriction (TAC) to create POH in C57/Bl6 mice. Experimental groups were sham, 1-week TAC (1wTAC) for early hypertrophy, or 6-week TAC (6wTAC) for established hypertrophy. We used western blots to determine O-GlcNAc regulation. To assess the effect of increased protein O-GlcNAcylation with established hypertrophy, mice received thiamet-g (TG) starting 4 weeks after TAC. Protein O-GlcNAc levels were significantly elevated in 1wTAC versus Sham with a fall in 6wTAC. OGA, which removes O-GlcNAc from proteins, fell in 1wTAC versus sham. GFAT is the rate-limiting HBP enzyme and the isoform GFAT1 substantially rose in 1wTAC. With established hypertrophy, TG increased protein O-GlcNAc levels but did not affect cardiac mass. In summary, protein O-GlcNAc levels vary during POH with elevations occurring during active hypertrophic growth early after TAC. O-GlcNAc levels appear to be regulated by changes in key enzyme levels. Increasing O-GlcNAc levels during established hypertrophy did not restart hypertrophic growth.

Objectives:. Effective treatment options for surfactant therapy in acute respiratory distress syndrome and coronavirus disease 2019 have not been established. To conduct preclinical studies in vitro and in vivo to evaluate efficiency, particle size, dosing, safety, and efficacy of inhaled surfactant using a breath-synchronized, nebulized delivery system in an established acute respiratory distress syndrome model. Design:. Preclinical study. Setting:. Research laboratory. Subjects:. Anesthetized pigs. Intervention:. In vitro analysis included particle size distribution and inhaled dose during simulated ventilation using a novel breath-synchronized nebulizer. Physiologic effects of inhaled aerosolized surfactant (treatment) were compared with aerosolized normal saline (control) in an adult porcine model (weight of 34.3 ± 0.6 kg) of severe acute respiratory distress syndrome (Pao2/Fio2 <100) with lung lavages and ventilator-induced lung injury during invasive ventilation. Measurements and Main Results:. Mass median aerosol diameter was 2.8 µm. In vitro dose delivered distal to the endotracheal tube during mechanical ventilation was 85% ± 5%. Nebulizers were functional up to 20 doses of 108 mg of surfactant. Surfactant-treated animals (n = 4) exhibited rapid improvement in oxygenation with nearly full recovery of Pao2/Fio2 (~300) and end-expiratory lung volumes with nominal dose less than 30 mg/kg of surfactant, whereas control subjects (n = 3) maintained Pao2/Fio2 less than 100 over 4.5 hours with reduced end-expiratory lung volume. There was notably greater surfactant phospholipid content and lower indicators of lung inflammation and pathologic lung injury in surfactant-treated pigs than controls. There were no peridosing complications associated with nebulized surfactant, but surfactant-treated animals had progressively higher airway resistance post treatment than controls with no differences in ventilation effects between the two groups. Conclusions:. Breath-synchronized, nebulized bovine surfactant appears to be a safe and feasible treatment option for use in coronavirus disease 2019 and other severe forms of acute respiratory distress syndrome.

Background Protein posttranslational modifications by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) increase with cardiac hypertrophy, yet the functional effects of these changes are incompletely understood. In other organs, O‐GlcNAc promotes adaptation to acute physiological stressors; however, prolonged O‐GlcNAc elevations are believed to be detrimental. We hypothesize that early O‐GlcNAcylation improves cardiac function during initial response to pressure overload hypertrophy, but that sustained elevations during established pathological hypertrophy negatively impact cardiac function by adversely affecting calcium handling proteins. Methods and Results Transverse aortic constriction or sham surgeries were performed on littermate controls or cardiac‐specific, inducible O‐GlcNAc transferase knockout (OGTKO) mice to reduce O‐GlcNAc levels. O‐GlcNAc transferase deficiency was induced at different times. To evaluate the initial response to pressure overload, OGTKO was completed preoperatively and mice were followed for 2 weeks post‐surgery. To assess prolonged O‐GlcNAcylation during established hypertrophy, OGTKO was performed starting 18 days after surgery and mice were followed until 6 weeks post‐surgery. In both groups, OGTKO with transverse aortic constriction caused significant left ventricular dysfunction. OGTKO did not affect levels of the calcium handling protein SERCA2a. OGTKO reduced phosphorylation of phospholamban and cardiac troponin I, which would negatively impact cardiac function. O‐GlcNAcylation of protein kinase A catalytic subunit, a kinase for phospholamban, decreased with OGTKO. Conclusions O‐GlcNAcylation promotes compensated cardiac function in both early and established pathological hypertrophy. We identified a novel O‐GlcNAcylation of protein kinase A catalytic subunit, which may regulate calcium handling and cardiac function.

Ischemia-reperfusion (I/R) results in altered metabolic and molecular responses, and phosphorylation is one of the most noted regulatory mechanisms mediating signaling mechanisms during physiological stresses. To expand our knowledge of the potential phosphoproteomic changes in the myocardium during I/R, we used Isobaric Tags for Relative and Absolute Quantitation-based analyses in left ventricular samples obtained from porcine hearts under control or I/R conditions. The data are available via ProteomeXchange with identifier PXD006066. We identified 1,896 phosphopeptides within left ventricular control and I/R porcine samples. Significant differential phosphorylation between control and I/R groups was discovered in 111 phosphopeptides from 86 proteins. Analysis of the phosphopeptides using Motif-x identified five motifs: (..R..S..), (..SP..), (..S.S..), (..S…S..), and (..S.T..). Semiquantitative immunoblots confirmed site location and directional changes in phosphorylation for phospholamban and pyruvate dehydrogenase E1, two proteins known to be altered by I/R and identified by this study. Novel phosphorylation sites associated with I/R were also identified. Functional characterization of the phosphopeptides identified by our methodology could expand our understanding of the signaling mechanisms involved during I/R damage in the heart as well as identify new areas to target therapeutic strategies.NEW & NOTEWORTHY We used Isobaric Tags for Relative and Absolute Quantitation technology to investigate the phosphoproteomic changes that occur in cardiac tissue under ischemia-reperfusion conditions. The results of this study provide an extensive catalog of phosphoproteins, both predicted and novel, associated with ischemia-reperfusion, thereby identifying new pathways for investigation.

Venoarterial extracorporeal membrane oxygenation (VA-ECMO) provides hemodynamic rescue for patients encountering right or left ventricular (RV or LV) decompensation, particularly after surgery for congenital heart defects. ECMO, supported metabolically by parenteral nutrition, provides reductions in myocardial work and energy demand and, therefore, enhances functional recovery. The RV must often assume systemic ventricular pressures and function on weaning from VA-ECMO. However the substrate utilization responses of the RV to VA-ECMO or stimulation are unknown. We determined RV and LV substrate utilization response to VA-ECMO in immature swine heart. Mixed-breed male Yorkshire pigs (33-49 days old) underwent normal pressure volume loading (control, n = 5) or were unloaded by VA-ECMO (ECMO, n = 10) for 8 h. Five pigs with ECMO received intravenous thyroid hormone [triiodothyronine (T3)] to alter substrate utilization. Carbon 13 (13C)-labeled substrates (lactate and medium-chain and long-chain fatty acids) were systemically infused as metabolic tracers. Analyses by nuclear magnetic resonance showed that both ventricles have similar trends of fractional 13C-labeled substrate contributions to the citric acid cycle under control conditions. VA-ECMO produced higher long-chain fatty acids and lower lactate contribution to the citric acid cycle via inhibition of pyruvate dehydrogenase, whereas T3 promoted lactate metabolism in both ventricles. However, these metabolic shifts were smaller in RV, and RV fatty acid contributions showed minimal response to perturbations. Furthermore, VA-ECMO and T3 also achieved high [phosphocreatine]/[ATP] and low [NADH]/[NAD+] in LV but not in RV. These data suggest that the RV shows decreased ability to modify substrate utilization and achieve improvements in energy supply/demand during VA-ECMO.NEW & NOTEWORTHY We showed that the right ventricle unloaded by venoarterial extracorporeal membrane oxygenation (VA-ECMO) has diminished capacity to alter substrate utilization compared with the left ventricle. This decrease in metabolic flexibility contributes to the inability to increase high-energy phosphate reserves during myocardial rest by VA-ECMO.

Deep hypothermic circulatory arrest is often required for the repair of complex congenital cardiac defects in infants. However, deep hypothermic circulatory arrest induces neuroapoptosis associated with later development of neurocognitive abnormalities. Selective cerebral perfusion theoretically provides superior neural protection possibly through modifications in cerebral substrate oxidation and closely integrated glutamate cycling. We tested the hypothesis that selective cerebral perfusion modulates glucose utilization, and ameliorates abnormalities in glutamate flux, which occur in association with neuroapoptosis during deep hypothermic circulatory arrest. Eighteen infant male Yorkshire piglets were assigned randomly to two groups of seven (deep hypothermic circulatory arrest or deep hypothermic circulatory arrest with selective cerebral perfusion for 60 minutes at 18?) and four control pigs without cardiopulmonary bypass support. Carbon-13-labeled glucose as a metabolic tracer was infused, and gas chromatography-mass spectrometry and nuclear magnetic resonance were used for metabolic analysis in the frontal cortex. Following 2.5h of cerebral reperfusion, we observed similar cerebral adenosine triphosphate levels, absolute levels of lactate and citric acid cycle intermediates, and carbon-13 enrichment among three groups. However, deep hypothermic circulatory arrest induced significant abnormalities in glutamate cycling resulting in reduced glutamate/glutamine and elevated -aminobutyric acid/glutamate along with neuroapoptosis, which were all prevented by selective cerebral perfusion. The data suggest that selective cerebral perfusion prevents these modifications in glutamate/glutamine/-aminobutyric acid cycling and protects the cerebral cortex from apoptosis.

Children with sepsis and multisystem organ failure have downregulated leukocyte gene expression of peroxisome proliferator-activated receptor-α (PPARα), a nuclear hormone receptor transcription factor that regulates inflammation and lipid metabolism. Mouse models of sepsis have likewise demonstrated that the absence of PPARα is associated with decreased survival and organ injury, specifically of the heart. Using a clinically relevant mouse model of early sepsis, we found that heart function increases in wild-type (WT) mice over the first 24 h of sepsis, but that mice lacking PPARα (Ppara-/-) cannot sustain the elevated heart function necessary to compensate for sepsis pathophysiology. Left ventricular shortening fraction, measured 24 h after initiation of sepsis by echocardiography, was higher in WT mice than in Ppara-/- mice. Ex vivo working heart studies demonstrated greater developed pressure, contractility, and aortic outflow in WT compared with Ppara-/- mice. Furthermore, cardiac fatty acid oxidation was increased in WT but not in Ppara-/- mice. Regulatory pathways controlling pyruvate incorporation into the citric acid cycle were inhibited by sepsis in both genotypes, but the regulatory state of enzymes controlling fatty acid oxidation appeared to be permissive in WT mice only. Mitochondrial ultrastructure was not altered in either genotype indicating that severe mitochondrial dysfunction is unlikely at this stage of sepsis. These data suggest that PPARα expression supports the hyperdynamic cardiac response early in the course of sepsis and that increased fatty acid oxidation may prevent morbidity and mortality. NEW & NOTEWORTHY:In contrast to previous studies in septic shock using experimental mouse models, we are the first to demonstrate that heart function increases early in sepsis with an associated augmentation of cardiac fatty acid oxidation. Absence of peroxisome proliferator-activated receptor-α (PPARα) results in reduced cardiac performance and fatty acid oxidation in sepsis.

Introduction: Complex congenital cardiac defect repair in infants often requires deep hypothermic circulatory arrest (DHCA). As immediate or late neurological complications still occur, adjunctive selective cerebral perfusion (SCP) theoretically provides superior neural protection by supplying oxygen and substrate and maintaining energy metabolism. Hypothesis: SCP preserves glucose utilization, energy metabolism, and glutamate cycling in comparison to DHCA alone. Methods: Fourteen male Yorkshire piglets (age 34-44 days) were assigned randomly to 2 groups (DHCA or SCP for 60 minutes at 18°C). Cerebral perfusion through 1st branch of aorta was maintained at 40 ml/kg/min during SCP. After the completion of rewarming, 13-Carbon-labeled glucose ( 13 C-glucose) was infused through common carotid artery for 1 hour under continuous cardiopulmonary bypass support and then cerebral tissue was extracted. Gas chromatography-mass spectrometry and nuclear magnetic resonance were used for cerebral metabolic analysis in the frontal cortex of brain. Results: There were no operative or technical complications in any groups. Cerebral rSO 2 in simple DHCA dropped substantially to ~ 25% during DHCA, but maintained over 80% in SCP. Apoptosis by TUNEL assay was significantly less in SCP at 2.5 hours after DHCA. ATP levels in the extracted cerebral tissue were similar between 2 groups but glycogen store were higher in SCP. Absolute levels of lactate and citric acid cycle intermediates and their 13 C-enrichment were similar in the cerebral tissue between the 2 groups. However, SCP increased absolute and 13 C-enrichment for glutamate and compared to DHCA, while gamma aminobutyric acid (GABA) increased in DHCA. Conclusions: SCP added to DHCA does not provide additional ATP preservation or modify glucose oxidation. However, SCP does alter glucose cycling into alternate pathways, leading to preservation of glycogen, and shifts flux away from GABA, a major inhibitory neurotransmitter, and towards glutamate. These flux changes suggest that SCP decreases synaptic inhibition, which may relate to the reduced neural apoptosis.

Hypertrophic stimuli cause transcription of the proto-oncogene c-Myc (Myc). Prior work showed that myocardial knockout of c-Myc (Myc) attenuated hypertrophy and decreased expression of metabolic genes after aortic constriction. Accordingly, we assessed the interplay between Myc, substrate oxidation and cardiac function during early pressure overload hypertrophy. Mice with cardiac specific, inducible Myc knockout (MycKO-TAC) and non-transgenic littermates (Cont-TAC) were subjected to transverse aortic constriction (TAC; n = 7/group). Additional groups underwent sham surgery (Cont-Sham and MycKO-Sham, n = 5 per group). After two weeks, function was measured in isolated working hearts along with substrate fractional contributions to the citric acid cycle by using perfusate with 13C labeled mixed fatty acids, lactate, ketone bodies and unlabeled glucose and insulin. Cardiac function was similar between groups after TAC although +dP/dT and -dP/dT trended towards improvement in MycKO-TAC versus Cont-TAC. In sham hearts, Myc knockout did not affect cardiac function or substrate preferences for the citric acid cycle. However, Myc knockout altered fractional contributions during TAC. The unlabeled fractional contribution increased in MycKO-TAC versus Cont-TAC, whereas ketone and free fatty acid fractional contributions decreased. Additionally, protein posttranslational modifications by O-GlcNAc were significantly greater in Cont-TAC versus both Cont-Sham and MycKO-TAC. In conclusion, Myc alters substrate preferences for the citric acid cycle during early pressure overload hypertrophy without negatively affecting cardiac function. Myc also affects protein posttranslational modifications by O-GlcNAc during hypertrophy, which may regulate Myc-induced metabolic changes.

Nutritional energy support during extracorporeal membrane oxygenation (ECMO) should promote successful myocardial adaptation and eventual weaning from the ECMO circuit. Fatty acids (FAs) are a major myocardial energy source, and medium-chain FAs (MCFAs) are easily taken up by cell and mitochondria without membrane transporters. Odd-numbered MCFAs supply carbons to the citric acid cycle (CAC) via anaplerotic propionyl-CoA as well as acetyl-CoA, the predominant β-oxidation product for even-numbered MCFA. Theoretically, this anaplerotic pathway enhances carbon entry into the CAC, and provides superior energy state and preservation of protein synthesis. We tested this hypothesis in an immature swine model undergoing ECMO. Fifteen male Yorkshire pigs (26-45 days old) with 8-h ECMO received either normal saline, heptanoate (odd-numbered MCFA), or octanoate (even-numbered MCFA) at 2.3 μmol·kg body wt(-1)·min(-1) as MCFAs systemically during ECMO (n = 5/group). The 13-carbon ((13)C)-labeled substrates ([2-(13)C]lactate, [5,6,7-(13)C3]heptanoate, and [U-(13)C6]leucine) were systemically infused as metabolic markers for the final 60 min before left ventricular tissue extraction. Extracted tissues were analyzed for the (13)C-labeled and absolute concentrations of metabolites by nuclear magnetic resonance and gas chromatography-mass spectrometry. Octanoate produced markedly higher myocardial citrate concentration, and led to a higher [ATP]-to-[ADP] ratio compared with other groups. Unexpectedly, octanoate and heptanoate increased the flux of propionyl-CoA relative to acetyl-CoA into the CAC compared with control. MCFAs promoted increases in leucine oxidation, but were not associated with a difference in protein synthesis rate. In conclusion, octanoate provides energetic advantages to the heart over heptanoate.

Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we tested the hypothesis that prolonged systemic pyruvate supplementation activates pyruvate oxidation in an immature swine model in vivo. Twelve male mixed-breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (group C) or pyruvate (group P) during the final 6 h of 8 h of ECMO. Over the final hour, piglets received [2-(13)C] pyruvate, as a reference substrate for oxidation, and [(13)C6]-l-leucine, as an indicator for amino acid oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of the citric acid cycle intermediates. An increase in anaplerotic flux through pyruvate carboxylation in group P occurred compared with no change in pyruvate oxidation. Additionally, pyruvate promoted an increase in the phosphorylation state of several nutrient-sensitive enzymes, like AMP-activated protein kinase and acetyl CoA carboxylase, suggesting activation for fatty acid oxidation. Pyruvate also promoted O-GlcNAcylation through the hexosamine biosynthetic pathway. In conclusion, although prolonged pyruvate supplementation did not alter pyruvate oxidation, it did elicit changes in nutrient- and energy-sensitive pathways. Therefore, the observed results support the further study of pyruvate and its downstream effect on cardiac function.

Background: Mouse echocardiography is an established, non-invasive method to evaluate experimental cardiovascular disease. The murine Kawasaki disease (KD) model demonstrates aortic and coronary artery inflammation by histology. The clinical relevance of these pathological changes has not been confirmed. In children, KD involves coronary artery abnormalities visualized by echocardiography. However, echocardiography has not been previously applied to the mouse KD model. We hypothesized that coronary vasculitis caused the lactobacillus casei cell wall extract (LCE) causes abnormalities detectable by echo. Methods: Male mice (ages 3-4 months) were injected with LCE or vehicle. Sedated echos were performed with a VisualSonics Vevo 2100 system. Twelve mice (8 LCE and 4 vehicle) had pre-injection echos followed by serial studies for 6-10 weeks. Evaluations were blinded to treatment group. 2-D measurements were taken at consistent locations in the arteries. Aortic regurgitation was rated based upon clinical criteria. Histology was evaluated at 6 weeks post-LCE. Results: Mild aortic regurgitation (AR) was present in 6 of 8 mice injected with LCE starting at 1 week post-injection. No AR was present at baseline or vehicle injected mice. LCE mice showed increased aortic root diameter at 6 weeks compared to baseline (1.78mm±0.01 vs 1.52±0.02, p<0.05). Overall left coronary artery dimensions were not changed from baseline at 6 weeks post-LCE, but coronary imaging was difficult. One mouse had a diffusely enlarged left coronary system and severely diminished left ventricular function at 10 weeks post-LCE. Histology findings were present in all LCE-injected mice including aortic valvulitis, myointimal proliferation and a single case of infarction. Conclusion: The most prominent echo findings in the mouse KD model are severe aortitis with dilation and valvular regurgitation. These features could serve as non-invasive experimental measurements. Coronary dilation was a rare finding; however the coronaries are difficult to evaluate in mice. Overall, the mouse KD model demonstrates greater aortic pathology than found in human KD. Further studies are underway to evaluate additional non-invasive measurements such as vascular strain.

Antifungal drugs have been proposed as a novel treatment for Acanthamoeba keratitis. The cysticidal activity of several antifungal compounds was tested against different genotypes of culture collection and clinical isolates of Acanthamoeba. Only voriconazole and posaconazole were found to be cysticidal, with no differences in activity observed between clinical and culture collection isolates.

Background: Extracorporeal membrane oxygenation (ECMO) is the most frequently used mechanical circulatory support in children with cardiopulmonary failure. Nutritional energy support as well as inotropic support is important to recover cardiac function during ECMO. Fatty acids (FAs) are major myocardial energy source for citric acid cycle (CAC). Medium-chain FAs (MCFAs) are easily uptaken into cell and mitochondria without membrane transporters. Our previous animal study suggested that MCFAs can potentially use for myocardial energy metabolism as the best accessible substrates during ECMO. Even-numbered MCFAs enter into CAC via acetyl-CoA, while odd-numbered MCFAs are anaplerotic for the CAC as propionyl-CoA in addition to acetyl-CoA. This study assessed whether odd- or even-numbered MCFAs product efficient energy during a short-term ECMO. Methods: Fifteen male Yorkshire pigs (26-45 days old) with 8-hour ECMO were received either normal saline, heptanoate (odd-numbered MCFA) or octanoate (even-numbered MCFA) at 2.3 μmol/kg body wt/min as MCFAs systemically during ECMO (n = 5 per group). The 13-Carbon ( 13 C) labeled substrates ([2- 13 C]lactate, [5,6,7- 13 C 3 ]heptanoate and [U- 13 C 6 ]leucine) as metabolic markers were infused into coronary artery for the final hour before left ventricular tissue extraction. Extracted left ventricular tissues were analyzed for the 13 C-labeled and absolute concentrations of metabolites by nuclear magnetic resonance and gas chromatography-mass spectrometry. Results: There were no adverse events in any groups during experiments. Octanoate produced markedly high citrate concentration, indicating the conjugation of CAC, and led to high [ATP]/[ADP] ratio compared with other groups (6.2 ± 0.6, 6.0 ± 0.6 and 7.8 ± 0.5 in normal saline, heptanoate and octanoate, P < 0.05). Octanoate strongly increased the CAC metabolism through anaplerotic pathway from propionyl-CoA, but not heptanoate. Lactate oxidation and protein synthesis ratio from leucine were not different among 3 groups. Conclusions: Even-numbered MCFAs was superior energy source for CAC than odd-numbered MCFAs in normal heart supported with ECMO. These results support that nutritional therapy with even-numbered MCFAs facilitates weaning from ECMO.

Background Extracorporeal membrane oxygenation ( ECMO ) provides a bridge to recovery after myocardial injury in infants and children, yet morbidity and mortality remain high. Weaning from the circuit requires adequate cardiac contractile function, which can be impaired by metabolic disturbances induced either by ischemia‐reperfusion and/or by ECMO . We tested the hypothesis that although ECMO partially ameliorates metabolic abnormalities induced by ischemia‐reperfusion , these abnormalities persist or recur with weaning. We also determined if thyroid hormone supplementation (triiodothyronine) during ECMO improves oxidative metabolism and cardiac function. Methods and Results Neonatal piglets underwent transient coronary ischemia to induce cardiac injury then were separated into 4 groups based on loading status. Piglets without coronary ischemia served as controls. We infused into the left coronary artery [2‐ 13 C]pyruvate and [ 13 C 6 , 15 N] l ‐leucine to evaluate oxidative metabolism by gas chromatography‐mass spectroscopy and nuclear magnetic resonance methods. ECMO improved survival, increased oxidative substrate contribution through pyruvate dehydrogenase, reduced succinate and fumarate accumulation, and ameliorated ATP depletion induced by ischemia. The functional and metabolic benefit of ECMO was lost with weaning, yet triiodothyronine supplementation during ECMO restored function, increased relative pyruvate dehydrogenase flux, reduced succinate and fumarate, and preserved ATP stores. Conclusions Although ECMO provides metabolic rest by decreasing energy demand, metabolic impairments persist, and are exacerbated with weaning. Treating ECMO ‐induced thyroid depression with triiodothyronine improves substrate flux, myocardial oxidative capacity and cardiac contractile function. This translational model suggests that metabolic targeting can improve weaning.