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Novel therapeutic approaches are needed for the treatment of Ewing sarcoma tumors. We previously identified that Ewing sarcoma cell lines are sensitive to drugs that inhibit protein translation. However, translational and therapeutic approaches to inhibit protein synthesis in tumors are limited. In this work, we identified that reactive oxygen species, which are generated by a wide range of chemotherapy and other drugs, inhibit protein synthesis and reduce the level of critical proteins that support tumorigenesis in Ewing sarcoma cells. In particular, we identified that both hydrogen peroxide and auranofin, an inhibitor of thioredoxin reductase and regulator of oxidative stress and reactive oxygen species, activate the repressor of protein translation 4E-BP1 and reduce the levels of the oncogenic proteins RRM2 and PLK1 in Ewing and other sarcoma cell lines. These results provide novel insight into the mechanism of how ROS-inducing drugs target cancer cells via inhibition of protein translation and identify a mechanistic link between ROS and the DNA replication (RRM2) and cell cycle regulatory (PLK1) pathways., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Haight, Koppenhafer, Geary and Gordon.)

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors have complicated the identification of the mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of the RRM1 protein results in the upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos, and downregulation of c-Myc. Notably, overexpression of c-Jun and c-Fos in Ewing sarcoma cells is sufficient to inhibit cell growth and downregulate the expression of the c-Myc oncogene. We also identified that the upregulation of AP-1 is mediated, in part, by SLFN11, which is a replication stress response protein that is expressed at high levels in Ewing sarcoma. In addition, small-molecule inhibitors of RNR, including gemcitabine, and histone deacetylase inhibitors, which reduce the level of the RRM1 protein, also activate AP-1 signaling and downregulate the level of c-Myc in Ewing sarcoma. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR., Significance: RNR is the rate-limiting enzyme in the synthesis of deoxyribonucleotides. Although RNR is the target of multiple chemotherapy drugs, polypharmacology and off-target effects have complicated the identification of the precise mechanism of action of these drugs. In this work, using a knockout-rescue approach, we identified that inhibition of RNR upregulates AP-1 signaling and downregulates the level of c-Myc in Ewing sarcoma tumors., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Sarcomas are difficult to treat and the therapy, even when effective, is associated with long-term and life-threatening side effects. In addition, the treatment regimens for many sarcomas, including Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma, are relatively unchanged over the past two decades, indicating a critical lack of progress. Although differentiation-based therapies are used for the treatment of some cancers, the application of this approach to sarcomas has proven challenging. Here, using a CRISPR-mediated gene knockout approach, we show that Inhibitor of DNA Binding 2 (ID2) is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo in Ewing sarcoma tumors. We also identified that homoharringtonine, which is an inhibitor of protein translation and FDA-approved for the treatment of leukemia, decreases the level of the ID2 protein and significantly reduces tumor growth and prolongs mouse survival in an Ewing sarcoma xenograft model. Furthermore, in addition to targeting ID2, homoharringtonine also reduces the protein levels of ID1 and ID3, which are additional members of the ID family of proteins with well-described roles in tumorigenesis, in multiple types of cancer. Overall, these results provide insight into developmental regulation in Ewing sarcoma tumors and identify a novel, therapeutic approach to target the ID family of proteins using an FDA-approved drug., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted along with NF1 , a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of MPNSTs is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown., Methods: Using CRISPR-Cas9 targeting of Nf1 + Cdkn2a or Nf1 + Tp53 in the mouse sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Terminal tumors were evaluated by western blot, qRT-PCR, and immunohistochemistry., Results: Mice lacking Rabl6 displayed slower tumor progression and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in Rabl6 -null, Nf1 + Cdkn2a lesions whereas loss of RABL6A caused upregulation of the CDK inhibitor, p27, in all tumors. Paradoxically, both models displayed elevated Myc protein and Ki67 staining in terminal tumors lacking RABL6A. In Nf1 + p53 tumors, cellular atypia and polyploidy were evident and increased by RABL6A loss., Conclusions: These findings demonstrate that RABL6A is required for optimal progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for unwanted alterations, including increased Myc, cellular atypia, and polyploidy, that ultimately promote a hyper-proliferative tumor phenotype akin to drug-resistant lesions., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Ribonucleotide reductase (RNR), which is a heterodimeric tetramer composed of RRM1 and RRM2 subunits, is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and essential for both DNA replication and the repair of DNA damage. The activity of RNR is coordinated with the cell cycle and regulated by fluctuations in the level of the RRM2 subunit. Multiple cancer types, including Ewing sarcoma tumors, are sensitive to inhibitors of RNR or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. Here, we show that the expression of the RRM2 protein is dependent on active protein synthesis and that 4E-BP1, a repressor of cap-dependent protein translation, specifically regulates the level of the RRM2 protein. Furthermore, inhibition of mTORC1/2, but not mTORC1, activates 4E-BP1, inhibits protein synthesis, and reduces the level of the RRM2 protein in multiple sarcoma cell lines. This effect of mTORC1/2 inhibitors on protein synthesis and RRM2 levels was rescued in cell lines with the CRISPR/Cas9-mediated knockout of 4E-BP1. In addition, the inducible expression of a mutant 4E-BP1 protein that cannot be phosphorylated by mTOR blocked protein synthesis and inhibited the growth of Ewing sarcoma cells in vitro and in vivo in a xenograft. Overall, these results provide insight into the multifaceted regulation of RRM2 protein levels and identify a regulatory link between protein translation and DNA replication.

Background: The treatment of Ewing sarcoma, an aggressive bone and soft tissue sarcoma, is associated with suboptimal outcomes and significant side-effects. Consequently, there is an urgent need to identify novel therapies that will improve outcomes for children and adults with Ewing sarcoma tumors while also decreasing treatment-related toxicities., Methods: We analyzed data from the PRISM drug repurposing screen, which tested the activity of 4518 drugs across 578 cancer cell lines, to identify drugs that selectively inhibit the growth of Ewing sarcoma cell lines. We then tested the effects of a top hit from the screen on cell proliferation, cell cycle progression, and activation of the DNA damage pathway using Ewing sarcoma cell lines. We also used a CRISPR/Cas9 gene knockout approach to investigate the role of Schlafen 11 (SLFN11), a restriction factor for DNA replication stress that is overexpressed in Ewing sarcoma tumors, in mediating the sensitivity of Ewing sarcoma cells to the drug., Results: We found that eltrombopag, an FDA-approved thrombopoietin-receptor agonist (TPO-RA) that is currently being evaluated as a treatment for chemotherapy-induced thrombocytopenia, inhibits the growth of Ewing sarcoma cell lines in vitro in proliferation and colony formation assays. However, from a mechanistic standpoint, the thrombopoietin receptor is not expressed in Ewing sarcoma cells and we show that eltrombopag impairs DNA replication and causes DNA damage in Ewing sarcoma cells by chelating iron, a known "off-target" effect of the drug. We also found that the sensitivity of Ewing sarcoma cells to eltrombopag is mediated, in part, by SLFN11, which regulates the cellular response to DNA replication stress., Conclusions: Ewing sarcoma cell lines are sensitive to eltrombopag and this drug could improve outcomes for patients with Ewing sarcoma tumors by both targeting the tumor, via chelation of iron and inhibition of DNA replication, and reducing chemotherapy-induced thrombocytopenia, via stimulation of the thrombopoietin receptor.

Inhibition of ribonucleotide reductase (RNR), the rate-limiting enzyme in the synthesis of deoxyribonucleotides, causes DNA replication stress and activates the ataxia telangiectasia and rad3-related protein (ATR)-checkpoint kinase 1 (CHK1) pathway. Notably, a number of different cancers, including Ewing sarcoma tumors, are sensitive to the combination of RNR and ATR-CHK1 inhibitors. However, multiple, overlapping mechanisms are reported to underlie the toxicity of ATR-CHK1 inhibitors, both as single agents and in combination with RNR inhibitors, toward cancer cells. Here, we identified a feedback loop in Ewing sarcoma cells in which inhibition of the ATR-CHK1 pathway depletes RRM2, the small subunit of RNR, and exacerbates the DNA replication stress and DNA damage caused by RNR inhibitors. Mechanistically, we identified that the inhibition of ATR-CHK1 activates CDK2, which targets RRM2 for degradation via the proteasome. Similarly, activation of CDK2 by inhibition or knockdown of the WEE1 kinase also depletes RRM2 and causes DNA damage and apoptosis. Moreover, we show that the concurrent inhibition of ATR and WEE1 has a synergistic effect in Ewing sarcoma cells. Overall, our results provide novel insight into the response to DNA replication stress, as well as a rationale for targeting the ATR, CHK1, and WEE1 pathways, in Ewing sarcoma tumors. IMPLICATIONS: Targeting the ATR, CHK1, and WEE1 kinases in Ewing sarcoma cells activates CDK2 and increases DNA replication stress by promoting the proteasome-mediated degradation of RRM2., (©2019 American Association for Cancer Research.)

The clinical potential of pharmacologic ascorbate (P-AscH - ; intravenous delivery achieving mmol/L concentrations in blood) as an adjuvant in cancer therapy is being reevaluated. At mmol/L concentrations, P-AscH - is thought to exhibit anticancer activity via generation of a flux of H 2 O 2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer to examine the effects of P-AscH - on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H 2 O 2 produced by P-AscH - induces DNA damage. In response to this DNA damage, we observed that PARP1 is hyperactivated. Using our unique absolute quantitation, we found that P-AscH - mediated the overactivation of PARP1, which results in consumption of NAD + , and subsequently depletion of ATP leading to mitotic cell death. We have also found that Chk1 plays a major role in the maintenance of genomic integrity following treatment with P-AscH - . Hyperactivation of PARP1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 analyzer, we demonstrated that the severe decrease in ATP after challenging with P-AscH - is because of increased demand, not changes in the rate of production. Genetic deletion and pharmacologic inhibition of PARP1 preserved both NAD + and ATP; however, the toxicity of P-AscH - remained. These data indicate that disruption of bioenergetics is a secondary factor in the toxicity of P-AscH - ; damage to DNA appears to be the primary factor. IMPLICATIONS: Efforts to leverage P-AscH - in cancer therapy should first focus on DNA damage., (©2019 American Association for Cancer Research.)

The treatment of Ewing sarcoma has changed very little in the past two decades and novel treatment approaches are needed. We recently identified that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), the rate-limiting enzyme in the synthesis of deoxyribonucleotides. We subsequently found that the inhibition of checkpoint kinase 1 (CHK1) increases the sensitivity of Ewing sarcoma cells to inhibitors of RNR, such as gemcitabine. However, Ewing sarcoma cells exhibit high levels of the CHK1 protein, which may represent an adaptive response to elevated levels of endogenous DNA replication stress. Consequently, we began this work with the aim of determining the impact of CHK1 levels on drug sensitivity, as well as identifying the mechanisms and pathways that regulate CHK1 levels in Ewing sarcoma cells. In this report, we show that the high levels of the CHK1 protein in Ewing sarcoma cells limit the efficacy of CHK1 inhibitors. However, inhibition of mTORC1/2 activates the translational repressor 4E-BP1, reduces protein synthesis, and decreases levels of the CHK1 protein in Ewing sarcoma cells. Similarly, we identified that the CHK1 inhibitor prexasertib also activates 4E-BP1, inhibits protein synthesis, and reduces CHK1 protein levels in Ewing sarcoma cells. Moreover, the combination of prexasertib and gemcitabine was synergistic in vitro , caused tumor regression in vivo , and significantly prolonged mouse survival in a Ewing sarcoma xenograft experiment. Overall, our results provide insight into Ewing sarcoma biology and support further investigation of the CHK1 pathway as a therapeutic target in Ewing sarcoma tumors., (©2018 American Association for Cancer Research.)

Ewing sarcoma is a bone and soft tissue sarcoma that occurs in children and young adults. The EWS-FLI1 gene fusion is the driver mutation in most Ewing sarcoma tumors and functions, in part, as an aberrant transcription factor. We recently identified that Ewing sarcoma cells are sensitive to inhibition of ribonucleotide reductase (RNR), which catalyzes the formation of deoxyribonucleotides from ribonucleotides. In this report, we show that Ewing sarcoma cells are sensitive to treatment with clofarabine, which is a nucleoside analogue and allosteric inhibitor of RNR. However, clofarabine is a reversible inhibitor of RNR and we found that the effect of clofarabine is limited when using a short (6-hour) drug treatment. Gemcitabine, on the other hand, is an irreversible inhibitor of the RRM1 subunit of RNR and this drug induces apoptosis in Ewing sarcoma cells when used in both 6-hour and longer drug treatments. Treatment of Ewing sarcoma cells with gemcitabine also results in activation of checkpoint kinase 1 (CHK1), which is a critical mediator of cell survival in the setting of impaired DNA replication. Notably, inhibition of CHK1 function in Ewing sarcoma cells using a small-molecule CHK1 inhibitor, or siRNA knockdown, in combination with gemcitabine results in increased toxicity both in vitro and in vivo in a mouse xenograft experiment. Overall, our results provide insight into Ewing sarcoma biology and identify a candidate therapeutic target, and drug combination, in Ewing sarcoma., Competing Interests: CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest.

Background: In utero exposure to hyperglycemia is becoming increasingly prevalent as the number of women entering pregnancy with type II diabetes, or developing gestational diabetes, increases. Both animal studies and epidemiologic investigations have found cardiovascular abnormalities in adult offspring of hyperglycemic mothers (OHM)., Objective: We hypothesized that adult OHM would have abnormal cardiac function in vivo and increased susceptibility to ischemia., Methods: Pregnant rats were made diabetic on day 12 of gestation. Serum glucose was monitored twice daily and insulin provided to maintain serum glucose at 200-400 mg/dl. Offspring were fostered to normal mothers after birth. Adult OHM were studied at 8-10 months of age with echocardiography to assess in vivo cardiac function and isolated hearts to determine the response to ischemia., Results: Echocardiography found significant diastolic dysfunction in male OHM compared to male controls. In isolated hearts, baseline cardiac function and left ventricular compliance was significantly diminished in male OHM compared to controls. Ischemia caused a significant decline in heart function in controls and female OHM, while function in male OHM remained unchanged., Conclusions: Adult male OHM demonstrate programmed cardiac dysfunction. Given the growing number of pregnancies complicated by hyperglycemia, additional assessment of cardiac function of adults born to diabetic mothers may be warranted., (Copyright © 2011 S. Karger AG, Basel.)

ODM (offspring of diabetic mothers) have an increased risk of developing metabolic and cardiovascular dysfunction; however, few studies have focused on the susceptibility to disease in offspring of mothers developing diabetes during pregnancy. We developed an animal model of late gestation diabetic pregnancy and characterized metabolic and vascular function in the offspring. Diabetes was induced by streptozotocin (50 mg/kg of body weight, intraperitoneally) in pregnant rats on gestational day 13 and was partially controlled by twice-daily injections of insulin. At 2 months of age, ODM had slightly better glucose tolerance than controls (P<0.05); however, by 6 months of age this trend had reversed. A euglycaemic-hyperinsulinamic clamp revealed insulin resistance in male ODM (P<0.05). In 6-8-month-old female ODM, aortas had significantly enhanced contractility in response to KCl, ET-1 (endothelin-1) and NA (noradrenaline). No differences in responses to ET-1 and NA were apparent with co-administration of L-NNA (NG-nitro-L-arginine). Relaxation in response to ACh (acetylcholine), but not SNP (sodium nitroprusside), was significantly impaired in female ODM. In contrast, males had no between-group differences in response to vasoconstrictors, whereas relaxation to SNP and ACh was greater in ODM compared with control animals. Thus the development of diabetes during pregnancy programmes gender-specific insulin resistance and vascular dysfunction in adult offspring.

Exposure to an adverse intrauterine environment is recognized as an important risk factor for the development of cardiovascular disease later in life. Although oxidative stress has been proposed as a mechanism for the fetal programming phenotype, the role of mitochondrial O(2)(*-) (superoxide radical) production has not been explored. To determine whether mitochondrial ROS (reactive oxygen species) production is altered by in utero programming, pregnant ewes were given a 48-h dexamethasone (dexamethasone-exposed, 0.28 mg.kg(-1) of body weight.day(-1)) or saline (control) infusion at 27-28 days gestation (term=145 days). Intact left ventricular mitochondria and freeze-thaw mitochondrial membranes were studied from offspring at 4-months of age. AmplexRed was used to measure H(2)O(2) production. Activities of the antioxidant enzymes Mn-SOD (manganese superoxide dismutase), GPx (glutathione peroxidase) and catalase were measured. Compared with controls, a significant increase in Complex I H(2)O(2) production was found in intact mitochondria from dexamethasone-exposed animals. The treatment differences in Complex I-driven H(2)O(2) production were not seen in mitochondrial membranes. Consistent changes in H(2)O(2) production from Complex III in programmed animals were not found. Despite the increase in H(2)O(2) production in intact mitochondria from programmed animals, dexamethasone exposure significantly increased mitochondrial catalase activity, whereas Mn-SOD and GPx activities were unchanged. The results of the present study point to an increase in the rate of release of H(2)O(2) from programmed mitochondria despite an increase in catalase activity. Greater mitochondrial H(2)O(2) release into the cell may play a role in the development of adult disease following exposure to an adverse intrauterine environment.

Polyglutamine (polygln) diseases are a group of inherited neurodegenerative disorders characterized by protein misfolding and aggregation. Here, we investigate the role in polygln disease of heat shock proteins (Hsps), the major class of molecular chaperones responsible for modulating protein folding in the cell. In transfected COS7 and PC12 neural cells, we show that Hsp40 and Hsp70 chaperones localize to intranuclear aggregates formed by either mutant ataxin-3, the disease protein in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), or an unrelated green fluorescent protein fusion protein containing expanded polygln. We further demonstrate that expression of expanded polygln protein elicits a stress response in cells as manifested by marked induction of Hsp70. Studies of SCA3/MJD disease brain confirm these findings, showing localization of Hsp40 and, less commonly, Hsp70 chaperones to intranuclear ataxin-3 aggregates. In transfected cells, overexpression of either of two Hsp40 chaperones, the DNAJ protein homologs HDJ-1 and HDJ-2, suppresses aggregation of truncated or full-length mutant ataxin-3. Finally, we extend these studies to a PC12 neural model of polygln toxicity in which we demonstrate that overexpression of HDJ-1 suppresses polygln aggregation with a parallel decrease in toxicity. These results suggest that expanded polygln protein induces a stress response and that specific molecular chaperones may aid the handling of misfolded or aggregated polygln protein in neurons. This study has therapeutic implications because it suggests that efforts to increase chaperone activity may prove beneficial in this class of diseases.

Spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), is one of at least eight inherited neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein. Here we present two lines of evidence implicating the ubiquitin-proteasome pathway in SCA3/MJD pathogenesis. First, studies of both human disease tissue and in vitro models showed redistribution of the 26S proteasome complex into polyglutamine aggregates. In neurons from SCA3/MJD brain, the proteasome localized to intranuclear inclusions containing the mutant protein, ataxin-3. In transfected cells, the proteasome redistributed into inclusions formed by three expanded polyglutamine proteins: a pathologic ataxin-3 fragment, full-length mutant ataxin-3 and an unrelated GFP-polyglutamine fusion protein. Inclusion formation by the full-length mutant ataxin-3 required nuclear localization of the protein and occurred within specific subnuclear structures recently implicated in the regulation of cell death, promyelocytic leukemia antigen oncogenic domains. In a second set of experiments, inhibitors of the proteasome caused a repeat length-dependent increase in aggregate formation, implying that the proteasome plays a direct role in suppressing polyglutamine aggregation in disease. These results support a central role for protein misfolding in the pathogenesis of SCA3/MJD and suggest that modulating proteasome activity is a potential approach to altering the progression of this and other polyglutamine diseases.

Developmental downregulation of the malate-aspartate shuttle has been observed in cardiac mitochondria. The goals of this study were to determine the time course of the postnatal decline and to identify potential regulatory sites by measuring steady-state myocardial mRNA and protein levels of the mitochondrial proteins involved in the shuttle. By use of isolated porcine cardiac mitochondria incubated with saturating concentrations of the cytosolic components of the malate-aspartate shuttle, shuttle capacity was found to decline by approximately 50% during the first 5 wk of life (from 921 +/- 48 to 531 +/- 53 nmol.min-1.mg protein-1). Mitochondrial aspartate aminotransferase mRNA levels were greater in adult than in newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase in adult cardiac tissue were 224% of levels in newborn tissue, whereas protein levels were 54% greater in adult myocardium. Aspartate/glutamate carrier protein levels were also greater in adult than in newborn tissue. mRNA and protein levels of the oxoglutarate/malate carrier were increased in newborn myocardium. It was concluded that 1) myocardial malate-aspartate shuttle capacity declines rapidly after birth, 2) divergence of mitochondrial malate dehydrogenase mRNA and protein levels during development suggests posttranscriptional regulation of this protein, and 3) the developmental decline in malate-aspartate shuttle capacity is regulated by decreased oxoglutarate/malate carrier gene expression.

The alpha-glycerophosphate (alpha-GP) shuttle has been shown to play a role in reducing equivalent transfer in neonatal cardiac mitochondria. In adult heart mitochondria, alpha-GP shuttle activity is not detectable. The goals of the current study were to define the time course of the age-dependent decline in alpha-GP shuttle capacity and to identify the enzymatic step(s) of the alpha-GP shuttle which are regulated during development. Intact mitochondria were isolated from porcine hearts of various ages and assayed for alpha-GP shuttle capacity. By 5 weeks of age, alpha-GP shuttle capacity had decreased by nearly 39%. The cytosolic step of the shuttle, catalysed by cytosolic alpha-glycerophosphate dehydrogenase (c alpha-GPDH), demonstrated a significant increase between 0-2-day-old animals and adults. Partial cDNA clones of porcine c alpha-GPDH and mitochondrial alpha-glycerophosphate dehydrogenase (m alpha-GPDH) were prepared and used to quantitate expression of these genes. Using mRNA isolated from neonatal and adult porcine myocardium, expression of the c alpha-GPDH was unchanged, while expression of the m alpha-GPDH gene was present in neonatal but absent in adult myocardium. These results demonstrate a rapid postnatal decline in myocardial alpha-GP shuttle capacity which appears to be regulated by a decline in m alpha-GPDH gene expression.

The metabolic demands of the newborn heart are met primarily by glucose and lactate. Mitochondria are impermeable to the NADH produced by these cytosolic reactions. The malate/aspartate and alpha-glycerophosphate (alpha-GP) shuttles provide two pathways to transport reducing equivalents into mitochondria. The goals of this study were to compare the capacity of these shuttles in newborn and adult cardiac mitochondria and to measure the maximal activity of the mitochondrial enzymes involved in these shuttles. Shuttle and enzyme capacities were measured in isolated mitochondria from the left and right ventricular free wall of 0-3-d-old and adult pig hearts. Malate/aspartate shuttle capacity was nearly three times greater in the newborn left ventricle compared with adult (newborn, 616 +/- 24; adult, 232 +/- 28 nmol/min/mg; mean +/- SEM; n = 8; p < 0.00001). The capacity of the malate/aspartate shuttle of the right ventricular free wall was greater than the left in the adult heart. Despite a decrease in malate/aspartate shuttle capacity, maximal activity of mitochondrial matrix enzymes involved in this pathway were increased in adult mitochondria. alpha-GP shuttle activity was absent in adult myocardium. Newborn left ventricular myocardium had significant alpha-GP shuttle activity (44 +/- 4 nmol/min/mg) due to enhanced flavin-linked mitochondrial alpha-GP dehydrogenase activity compared with adult. Interventricular differences in the alpha-GP shuttle capacity were not found in newborn or adult hearts. These findings suggest a mechanism for the substrate preference of neonatal myocardium.

Quantitation of myocardial perfusion is feasible using contrast enhanced magnetic resonance imaging. A method to quantitate myocardial blood flow is provided by the Kety model modified to account for a diffusable tracer such as gadoteridol. In the present study, perfusion parameters of the modified Kety model (partition coefficient and extraction efficiency) were determined for gadoteridol in intact myocardium using a constant flow, isolated, perfused heart model. Perfusion conditions included hearts with normal perfusion, hearts made globally ischemic for 20 min then perfused normally, and hearts whose coronary flow was more than doubled with 9 microM adenosine. T1 relaxation times were rapidly measured at 0.5 T following step increases in perfusate gadoteridol concentration and at steady state. Both the partition coefficient and extraction efficiency were found to be significantly increased in ischemic/reperfused hearts compared to normal. While flow rates in adenosine hearts were too high for accurate extraction efficiency determination using this technique, the partition coefficient was no different between adenosine and normally perfused hearts. The method described in this article allowed the kinetic parameters of the modified Kety model to be determined in intact heart using NMR relaxation time measurements as the basis of the calculation.

To study the potential role of endothelin-1, a potent endothelium-derived vasoconstrictor peptide, in the pathophysiology of persistent pulmonary hypertension of the newborn (PPHN), we measured arterial concentrations of immunoreactive endothelin-1 (irET-1) in 24 neonates with PPHN. Secondary diagnoses included meconium aspiration syndrome (13 patients), sepsis (2), congenital diaphragmatic hernia (1), asphyxia (1), pulmonary hemorrhage (1), aspiration of blood (1), and respiratory distress syndrome (1). Compared with irET-1 levels in umbilical cord blood in normal infants (15.1 +/- 4.1 pg/ml; mean +/- SEM) and in newborn infants with hyaline membrane disease who were supported by mechanical ventilation (11.8 +/- 1.2 pg/ml), infants with PPHN had markedly elevated circulating irET-1 levels (27.6 +/- 3.6 pg/ml; p < 0.01 vs cord blood, hyaline membrane disease). Infants with severe PPHN requiring extracorporeal membrane oxygenation (ECMO) therapy had higher irET-1 levels than infants with milder disease (31.0 +/- 4.7 for ECMO-treated infants vs 21.2 +/- 2.0 for non-ECMO-treated infants; p < 0.05). In patients treated without ECMO, irET-1 progressively decreased during the following 3 to 5 days, paralleling clinical improvement. In contrast, irET-1 concentrations remained elevated in infants with severe PPHN during ECMO therapy. We conclude that circulating irET-1 levels are elevated in newborn infants with PPHN, are positively correlated with disease severity, and decline with resolution of disease in patients who do not require ECMO therapy. Whether endothelin-1 contributes directly to the pathophysiology of PPHN or is simply a marker of disease activity remains speculative.