Evaluation of lung volumes and gas exchange in surfactant-deficient rabbits between variable and fixed servo pressures during high-frequency jet ventilation.

OBJECTIVE: To investigate a novel servo pressure (SP) setting during high-frequency jet ventilation (HFJV) for a lung protective strategy in a neonatal model of acute respiratory distress. STUDY DESIGN: Comparison of efficacy between variable (standard) and fixed SP settings in a randomized animal study using rabbits (n = 10, mean weight = 1.80 kg) with surfactant deficiency by repeated lung lavages. RESULTS: Rabbits in the fixed SP group had greater peak inspiratory pressure, SP, minute volume, pH, and PaO2, and lower PaCO2 after lung lavage than the variable SP group. Lung volume monitoring with electrical impedance tomography showed that fixed SP reduced the decline of the global lung tidal variation at 30 min after lung lavage (-17.4% from baseline before lavage) compared to variable SP (-44.9%). CONCLUSION: HFJV with fixed SP significantly improved gas exchange and lung volumes compared to variable SP. Applying a fixed SP may have important clinical implications for patients receiving HFJV.

Evaluation of a Novel Dry Powder Surfactant Aerosol Delivery System for Use in Premature Infants Supported with Bubble CPAP.

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.

Right ventricular energy metabolism in a porcine model of acute right ventricular pressure overload after weaning from cardiopulmonary bypass.

Acute right ventricular pressure overload (RVPO) occurs following congenital heart surgery and often results in low cardiac output syndrome. We tested the hypothesis that the RV exhibits limited ability to modify substrate utilization in response to increasing energy requirements during acute RVPO after cardiopulmonary bypass (CPB). We assessed the RV fractional contributions (Fc) of substrates to the citric acid cycle in juvenile pigs exposed to acute RVPO by pulmonary artery banding (PAB) and CPB. Sixteen Yorkshire male pigs (median 38 days old, 12.2 kg of body weight) were randomized to SHAM (Ctrl, n = 5), 2-h CPB (CPB, n = 5) or CPB with PAB (PAB-CPB, n = 6). Carbon-13 (13 C)-labeled lactate, medium-chain, and mixed long-chain fatty acids (MCFA and LCFAs) were infused as metabolic tracers for energy substrates. After weaning from CPB, RV systolic pressure (RVSP) doubled baseline in PAB-CPB while piglets in CPB group maintained normal RVSP. Fc-LCFAs decreased significantly in order PAB-CPB > CPB > Ctrl groups by 13 C-NMR. Fc-lactate and Fc-MCFA were similar among the three groups. Intragroup analysis for PAB-CPB showed that the limited Fc-LCFAs appeared prominently in piglets exposed to high RVSP-to-left ventricular systolic pressure ratio and high RV rate-pressure product, an indicator of myocardial oxygen demand. Acute RVPO after CPB strongly inhibits LCFA oxidation without compensation by lactate oxidation, resulting in energy deficiency as determined by lower (phosphocreatine)/(adenosine triphosphate) in PAB-CPB. Adequate energy supply but also metabolic interventions may be required to circumvent these RV energy metabolic abnormalities during RVPO after CPB.

Characterization of main pulmonary artery and valve annulus region of piglets using echocardiography, uniaxial tensile testing, and a novel non-destructive technique.

Characterization of cardiovascular tissue geometry and mechanical properties of large animal models is essential when developing cardiovascular devices such as heart valve replacements. These datasets are especially critical when designing devices for pediatric patient populations, as there is often limited data for guidance. Here, we present a previously unavailable dataset capturing anatomical measurements and mechanical properties of juvenile Yorkshire (YO) and Yucatan (YU) porcine main pulmonary artery (PA) and pulmonary valve (PV) tissue regions that will inform pediatric heart valve design requirements for preclinical animal studies. In addition, we developed a novel radial balloon catheter-based method to measure tissue stiffness and validated it against a traditional uniaxial tensile testing method. YU piglets, which were significantly lower weight than YO counterparts despite similar age, had smaller PA and PV diameters (7.6-9.9 mm vs. 10.1-12.8 mm). Young's modulus (stiffness) was measured for the PA and the PV region using both the radial and uniaxial testing methods. There was no significant difference between the two breeds for Young's modulus measured in the elastic (YU PA 84.7 ± 37.3 kPa, YO PA 79.3 ± 15.7 kPa) and fibrous regimes (YU PA 308.6 ± 59.4 kPa, YO PA 355.7 ± 68.9 kPa) of the stress-strain curves. The two testing techniques also produced similar stiffness measurements for the PA and PV region, although PV data showed greater variation between techniques. Overall, YU and YO piglets had similar PA and PV diameters and tissue stiffness to previously reported infant pediatric patients. These results provide a previously unavailable age-specific juvenile porcine tissue geometry and stiffness dataset critical to the development of pediatric cardiovascular prostheses. Additionally, the data demonstrates the efficacy of a novel balloon catheter-based technique that could be adapted to non-destructively measure tissue stiffness in situ.

Transcutaneous ultrasound-mediated gene delivery into canine livers achieves therapeutic levels of factor VIII expression.

A safe, effective, and inclusive gene therapy will significantly benefit a large population of patients with hemophilia. We used a minimally invasive transcutaneous ultrasound-mediated gene delivery (UMGD) strategy combined with microbubbles (MBs) to enhance gene transfer into 4 canine livers. A mixture of high-expressing, liver-specific human factor VIII (hFVIII) plasmid and MBs was injected into the hepatic vein via balloon catheter under fluoroscopy guidance with simultaneous transcutaneous UMGD treatment targeting a specific liver lobe. Therapeutic levels of hFVIII expression were achieved in all 4 dogs, and hFVIII levels were maintained at a detectable level in 3 dogs throughout the 60-day experimental period. Plasmid copy numbers correlated with hFVIII antigen levels, and plasmid-derived messenger RNA (mRNA) was detected in treated livers. Liver transaminase levels and histology analysis indicated minimal liver damage and a rapid recovery after treatment. These results indicate that liver-targeted transcutaneous UMGD is promising as a clinically feasible therapy for hemophilia A and other diseases.

Physiologic Effects of Instilled and Aerosolized Surfactant Using a Breath-Synchronized Nebulizer on Surfactant-Deficient Rabbits.

Surfactant administration incorporates liquid bolus instillation via endotracheal tube catheter and use of a mechanical ventilator. Aerosolized surfactant has generated interest and conflicting data related to dose requirements and efficacy. We hypothesized that aerosolized surfactant with a novel breath-actuated vibrating mesh nebulizer would have similar efficacy and safety as instilled surfactant. Juvenile rabbits (1.50 ± 0.20 kg, n = 17) were sedated, anesthetized, intubated, and surfactant was depleted via lung lavage on mechanical ventilation. Subjects were randomized to receive standard dose liquid instillation via catheter (n = 5); low dose surfactant (n = 5) and standard dose surfactant (n = 5) via aerosol; and descriptive controls (no treatment, n = 2). Peridosing events, disease severity and gas exchange, were recorded every 30 min for 3 h following surfactant administration. Direct-Instillation group had higher incidence for peridosing events than aerosol. Standard dose liquid and aerosol groups had greater PaO2 from pre-treatment baseline following surfactant (p < 0.05) with greater ventilation efficiency with aerosol (p < 0.05). Our study showed similar improvement in oxygenation response with greater ventilation efficiency with aerosol than liquid bolus administration at the same dose with fewer peridosing events. Breath-synchronized aerosol via nebulizer has potential as a safe, effective, and economical alternative to bolus liquid surfactant instillation.

Breath-Synchronized Nebulized Surfactant in a Porcine Model of Acute Respiratory Distress Syndrome.

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.

Inhaled nitric oxide reduces injury and microglia activation in porcine hippocampus after deep hypothermic circulatory arrest.

OBJECTIVE: Dysregulation of local nitric oxide (NO) synthetases occurs during ischemia and reperfusion associated with cardiopulmonary bypass, deep hypothermic circulatory arrest (DHCA), and reperfusion. Rapid fluctuations in local NO occurring in neonates and infants probably contribute to inflammation-induced microglial activation and neuronal degeneration after these procedures, eventually impairing neurodevelopment. We evaluated the anti-inflammatory efficacy of inhaled NO (iNO) in a piglet model emulating conditions during pediatric open-heart surgery with DHCA. METHODS: Infant Yorkshire piglets underwent DHCA (18°C) for 30 minutes, followed by reperfusion and rewarming either with or without iNO (20 ppm) in the ventilator at the onset of reperfusion for 3 hours (n = 5 per group, DHCA-iNO and DHCA). Through craniotomy, brains were extracted after perfusion fixation for histology. RESULTS: Plasma NO metabolites were elevated 2.5 times baseline data before DHCA by iNO. Fluoro-Jade C staining identified significantly lower number of degenerating neurons in the hippocampus of the DHCA-iNO group (P = .02) compared with the DHCA group. Morphologic analyses of ionized calcium-binding adapter molecule-1 stained microglia, evaluating cell body and dendritic process geometry with Imaris imaging software, revealed subjectively less microglial activation in the hippocampus of pigs receiving iNO. CONCLUSIONS: Using DHCA for 30 minutes, consistent with clinical exposure, we noted that iNO reduces neuronal degeneration in the hippocampus. In addition, iNO reduces microglial activation in the hippocampus after DHCA. The data suggest that iNO reduces neuronal degeneration by ameliorating inflammation and may be a practical mode of neuroprotection for infants undergoing DHCA.

Shear stress associated with cardiopulmonary bypass induces expression of inflammatory cytokines and necroptosis in monocytes.

Cardiopulmonary bypass (CPB) is required during most cardiac surgeries. CBP drives systemic inflammation and multiorgan dysfunction that is especially severe in neonatal patients. Limited understanding of molecular mechanisms underlying CPB-associated inflammation presents a significant barrier to improve clinical outcomes. To better understand these clinical issues, we performed mRNA sequencing on total circulating leukocytes from neonatal patients undergoing CPB. Our data identify myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, IL-8 and TNF-α were inflammatory cytokines robustly upregulated in leukocytes from both patients and piglets exposed to CPB. To delineate the molecular mechanism, we exposed THP-1 human monocytic cells to CPB-like conditions, including artificial surfaces, high shear stress, and cooling/rewarming. Shear stress was found to drive cytokine upregulation via calcium-dependent signaling pathways. We also observed that a subpopulation of THP-1 cells died via TNF-α-mediated necroptosis, which we hypothesize contributes to post-CPB inflammation. Our study identifies a shear stress-modulated molecular mechanism that drives systemic inflammation in pediatric CPB patients. These are also the first data to our knowledge to demonstrate that shear stress causes necroptosis. Finally, we observe that calcium and TNF-α signaling are potentially novel targets to ameliorate post-CPB inflammation.

The effect of oral triiodothyronine supplementation on lactate and pyruvate after paediatric cardiac surgery.

OBJECTIVE: To determine if triiodothyronine alters lactate, glucose, and pyruvate metabolism, and if serum pyruvate concentration could serve as a predictor of low cardiac output syndrome in children after cardiopulmonary bypass procedures. METHODS: This study was ancillary to the Oral Triiodothyronine for Infants and Children undergoing Cardiopulmonary bypass (OTICC) trial. Serum pyruvate was measured in the first 48 patients and lactate and glucose were measured in all 208 patients enrolled in the OTICC study on the induction of anaesthesia, 1 and 24 hours post-aortic cross-clamp removal. Patients were also defined as having low cardiac output syndrome according to the OTICC trial protocol. RESULT: Amongst the designated patient population for pyruvate analysis, 22 received placebo, and 26 received triiodothyronine (T3). Lactate concentrations were nearly 20 times greater than pyruvate. Lactate and pyruvate levels were not significantly different between T3 and placebo group. Glucose levels were significantly higher in the placebo group mainly at 24-hour post-cross-clamp removal. Additionally, lactate and glucose levels peaked at 1-hour post-cross-clamp removal in low cardiac output syndrome and non-low cardiac output syndrome patients, but subsequently decreased at a slower rate in low cardiac output syndrome. Lactate and pyruvate concentrations correlated with glucose only prior to surgery. CONCLUSION: Thyroid supplementation does not alter systemic lactate/pyruvate metabolism after cardiopulmonary bypass and reperfusion. Pyruvate levels are not useful for predicting low cardiac output syndrome. Increased blood glucose may be regarded as a response to hypermetabolic stress, seen mostly in patients with low cardiac output syndrome.

Transcutaneous Ultrasound-Mediated Nonviral Gene Delivery to the Liver in a Porcine Model.

Ultrasound (US)-mediated gene delivery (UMGD) of nonviral vectors was demonstrated in this study to be an effective method to transfer genes into the livers of large animals via a minimally invasive approach. We developed a transhepatic venous nonviral gene delivery protocol in combination with transcutaneous, therapeutic US (tUS) to facilitate significant gene transfer in pig livers. A balloon catheter was inserted into the pig hepatic veins of the target liver lobes via jugular vein access under fluoroscopic guidance. tUS exposure was continuously applied to the lobe with simultaneous infusion of pGL4 plasmid (encoding a luciferase reporter gene) and microbubbles. tUS was delivered via an unfocused, two-element disc transducer (H105) or a novel focused, single-element transducer (H114). We found applying transcutaneous US using H114 and H105 with longer pulses and reduced acoustic pressures resulted in an over 100-fold increase in luciferase activity relative to untreated lobes. We also showed effective UMGD by achieving focal regions of >105 relative light units (RLUs)/mg protein with minimal tissue damage, demonstrating the feasibility for clinical translation of this technique to treat patients with genetic diseases.

Metabolic Response to Stress by the Immature Right Ventricle Exposed to Chronic Pressure Overload.

Background The right ventricle exposed to chronic pressure overload exhibits hypertrophy and decompensates when exposed to stress. We hypothesize that impaired ability to increase myocardial oxidative flux through pyruvate dehydrogenase leads to hypertrophied right ventricular (RV) dysfunction when exposed to hemodynamic stress, and pyruvate dehydrogenase stimulation can improve RV function. Methods and Results Infant male Yorkshire piglets (13.5±0.6 kg weight, n=19) were used to assess substrate fractional contribution to the citric acid cycle after sustained pulmonary artery banding (PAB). Carbon 13-labeled glucose, lactate, and leucine, oxidative substrate tracers for the citric acid cycle, were infused into the right coronary artery on 7 to 10 days after PAB. RV systolic pressure, RV free wall thickness, and individual cardiomyocyte cell size after PAB were significantly elevated compared with the sham group. Both fractional glucose and lactate oxidations in the PAB group were >2-fold higher than in the sham group. Pigs with overdrive atrial pacing (≈80% increase in heart rate) stress after PAB showed only a 22% increase in rate-pressure product from baseline before atrial pacing and limited carbohydrate oxidation rate in the right ventricle. Intracoronary infusion of dichloroacetate, a pyruvate dehydrogenase agonist, produced higher rate-pressure product (59% increase) in response to increased workload by atrial pacing in association with a marked increase in lactate oxidation. Conclusions The immature hypertrophied right ventricle shows limited ability to increase carbohydrate oxidation in response to tachycardia stress leading to energy supply/utilization imbalance and decreased systolic function. Enhanced pyruvate dehydrogenase activation by dichloroacetate increases energy supply and preserves hypertrophied RV contractile function during hemodynamic stress.

Metabolic Response of the Immature Right Ventricle to Acute Pressure Overloading.

BACKGROUND: Surgical palliation or repair of complex congenital heart disease in early infancy can produce right ventricular (RV) pressure overload, often leading to acute hemodynamic decompensation. The mechanisms causing this acute RV dysfunction remain unclear. We tested the hypothesis that the immature right ventricle lacks the ability to modify substrate metabolism in order to meet increased energy demands induced by acute pressure overloading. METHODS AND RESULTS: Twenty-two infant male mixed breed Yorkshire piglets were randomized to a sham operation (Control) or pulmonary artery banding yielding >2-fold elevation over baseline RV systolic pressure. We used carbon 13 (13C)-labeled substrates and proton nuclear magnetic resonance to assess RV energy metabolism. [Phosphocreatine]/[ATP] was significantly lower after pulmonary artery banding. [Phosphocreatine]/[ATP] inversely correlated with energy demand indexed by maximal sustained RV systolic pressure/left ventricular systolic pressure. Fractional contributions of fatty acids to citric acid cycle were significantly lower in the pulmonary artery banding group than in the Control group (medium-chain fatty acids; 14.5±1.6 versus 8.2±1.0%, long-chain fatty acids; 9.3±1.5 versus 5.1±1.1%). 13C-flux analysis showed that flux via pyruvate decarboxylation did not increase during RV pressure overloading. CONCLUSIONS: Acute RV pressure overload yielded a decrease in [phosphocreatine]/[ATP] ratio, implying that ATP production did not balance the increasing ATP requirement. Relative fatty acids oxidation decreased without a reciprocal increase in pyruvate decarboxylation. The data imply that RV inability to adjust substrate oxidation contributes to energy imbalance, and potentially to contractile failure. The data suggest that interventions directed at increasing RV pyruvate decarboxylation flux could ameliorate contractile dysfunction associated with acute pressure overloading.

Quantitative cardiac phosphoproteomics profiling during ischemia-reperfusion in an immature swine model.

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.

Right ventricular metabolism during venoarterial extracorporeal membrane oxygenation in immature swine heart in vivo.

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.

Selective cerebral perfusion prevents abnormalities in glutamate cycling and neuronal apoptosis in a model of infant deep hypothermic circulatory arrest and reperfusion.

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 2.5 h 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 γ-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/γ-aminobutyric acid cycling and protects the cerebral cortex from apoptosis.

c-Myc Alters Substrate Utilization and O-GlcNAc Protein Posttranslational Modifications without Altering Cardiac Function during Early Aortic Constriction.

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.

Differential effects of octanoate and heptanoate on myocardial metabolism during extracorporeal membrane oxygenation in an infant swine model.

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 ß-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.

Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model.

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.

Triiodothyronine activates lactate oxidation without impairing fatty acid oxidation and improves weaning from extracorporeal membrane oxygenation.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) provides a rescue for children with severe cardiac failure. It has previously been shown that triiodothyronine (T3) improves cardiac function by modulating pyruvate oxidation during weaning. This study focused on fatty acid (FA) metabolism modulated by T3 for weaning from ECMO after cardiac injury. METHODS AND RESULTS: Nineteen immature piglets (9.1-15.3 kg) were separated into 3 groups with ECMO (6.5 h) and wean: normal circulation (Group-C); transient coronary occlusion (10 min) for ischemia-reperfusion (IR) followed by ECMO (Group-IR); and IR with T3 supplementation (Group-IR-T3). 13-Carbon ((13)C)-labeled lactate, medium-chain and long-chain FAs, was infused as oxidative substrates. Substrate fractional contribution (FC) to the citric acid cycle was analyzed by(13)C-nuclear magnetic resonance. ECMO depressed circulating T3 levels to 40% of the baseline at 4 h and were restored in Group-IR-T3. Group-IR decreased cardiac power, which was not fully restorable and 2 pigs were lost because of weaning failure. Group-IR also depressed FC-lactate, while the excellent contractile function and energy efficiency in Group-IR-T3 occurred along with a marked FC-lactate increase and [adenosine triphosphate]/[adenosine diphosphate] without either decreasing FC-FAs or elevating myocardial oxygen consumption over Group-C or -IR. CONCLUSIONS: T3 releases inhibition of lactate oxidation following IR injury without impairing FA oxidation. These findings indicate that T3 depression during ECMO is maladaptive, and that restoring levels improves metabolic flux and enhances contractile function during weaning.