Background: Despite improvements in surgical techniques, ischemia-reperfusion injury (IR) with reduced ventricular functional recovery remains a major predictor of morbidity and mortality among newborns with congenital heart disease (CHD). Modulation of energy substrate metabolism positively affects IR in adult patients undergoing cardiac surgery. However, these findings cannot be extrapolated to newborns with CHD who may undergo obligatory maturational changes. We sought to determine age-related changes in major fatty acid (FA) and carbohydrate metabolism enzymes and their regulation at transcriptional level in human newborns with CHD undergoing corrective surgery. We also determined the difference in maturational changes in patients displaying hypertrophy and its correlation with functional recovery capacity. Methods: Right ventricular specimens were collected from 59 newborns (age 0-200 days) undergoing surgery. Based on a z-score for right ventricular free wall thickness, patients were stratified as non-hypertrophied (34) or hypertrophied (25). Western blot analysis for major FA and glucose metabolism enzymes, and transcriptional regulatory proteins was performed. Clinical outcomes including post-surgical right and left ventricular functional recovery were also assessed. Results: Since shortly after birth, neonates displayed an age-related increase in of FA oxidation, and a decrease in carbohydrate oxidative capacity. Hypertrophied hearts displayed an overall reduced expression of key proteins involved in the transcriptional regulation of cardiac energy metabolism at the mitochondrial level, in this way suggesting a vulnerable energetic state. In support with this hypothesis, following surgery, patients displaying hypertrophy presented with a worse ventricular functional recovery. The effects of hypertrophy on clinical outcomes remained significant when entered into multivariate analysis. Conclusions: In line with animal studies, human newborns with CHD undergo obligatory age-related metabolic maturational changes that are modulated in the setting of hypertrophic response and can be responsible for worse peri-procedural outcomes.

ABSTRACT Two pathways have been identified for peroxisome formation: (i) growth and division and (ii) de novo synthesis. Recent experiments determined that peroxisomes originate at the endoplasmic reticulum (ER). Although many proteins have been implicated in the peroxisome biogenic program, no proteins in the eukaryotic secretory pathway have been identified as having roles in peroxisome formation. Using the yeast Saccharomyces cerevisiae regulatable Tet promoter Hughes clone collection, we found that repression of the ER-associated secretory proteins Sec20p and Sec39p resulted in mislocalization of the peroxisomal matrix protein chimera Pot1p-green fluorescent protein (GFP) to the cytosol. Likewise, the peroxisomal membrane protein chimera Pex3p-GFP localized to tubular-vesicular structures in cells suppressed for Sec20p, Sec39p, and Dsl1p, which form a complex at the ER. Loss of Sec39p attenuated formation of Pex3p-derived peroxisomal structures following galactose induction of Pex3p-GFP expression from the GAL1 promoter. Expression of Sec20p, Sec39p, and Dsl1p was moderately increased in yeast grown under conditions that proliferate peroxisomes, and Sec20p, Sec39p, and Dsl1p were found to cofractionate with peroxisomes and colocalize with Pex3p-monomeric red fluorescent protein under these conditions. Our results show that SEC20 , SEC39 , and DSL1 are essential secretory genes involved in the early stages of peroxisome assembly, and this work is the first to identify and characterize an ER-associated secretory machinery involved in peroxisome biogenesis.

Oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4; also designated OSBP2 and HLM) are implicated in sterol-transport and/or sensing via binding to protein partners. The aggregation of vimentin by an N-terminal-truncated variant of ORP4 (ORP4S), but not full-length ORP4L, suggested a functional interaction with this intermediate filament. Herein, we identify ORP4 domains that interact with vimentin, and determine how sterols and OSBP influence this activity. In CHO cells, ORP4L co-localized with filamentous vimentin but extensive remodeling of vimentin filaments required mutation of a leucine repeat motif (amino acids 361–382) adjacent to the oxysterol-binding domain. Similarly, the absence of the leucine repeat in ORP4S 418–878 resulted in co-localization with aggregated vimentin filaments, suggesting that both the sterol-binding domain and leucine repeat are involved. Transient expression of OSBP leucine repeat mutants also promoted vimentin aggregation by a mechanism involving heterodimerization with ORP4L. Glutathione S-transferase (GST)–ORP4 380–878 bound vimentin, cholesterol and 25-hydroxycholesterol in vitro. However, sterol-binding or a mutation that ablated sterol-binding did not influence the interaction of GST–ORP4 with vimentin. Thus the sterol-binding domain of ORP4 binds vimentin, cholesterol and oxysterols, and interacts with the filamentous vimentin network.

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.

De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi-ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.

The cytotoxic effects of several chemotherapeutic drugs have been linked to elevated de novo ceramide biosynthesis. However, the relationship between the intracellular site(s) of ceramide accumulation and cytotoxicity is poorly understood. Here we examined the relationship between the site of ceramide deposition and inhibition of protein translation and induction of apoptosis by the antitumor/antiviral xanthate, D609. In Chinese hamster ovary (CHO)-K1, HEK-293, and NIH-3T3 cells, D609 caused rapid (1-5 min) and sustained eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation followed by apoptosis after 24 h. Concurrently, D609 stimulated de novo ceramide synthesis and increased ceramide mass 2-fold by 2 h in CHO-K1 cells. In D609-treated CHO-K1 cells, sphingomyelin synthesis was stimulated by brefeldin A, and C-5-DMB-ceramide transport to the Golgi apparatus was blocked, indicating ceramide accumulation in the endoplasmic reticulum (ER). However, D609-mediated eIF2alpha phosphorylation, inhibition of protein synthesis, and apoptosis in CHO-K1 cells were not attenuated by fumonisin III or L-cycloserine. Interestingly, short-chain ceramide promoted eIF2alpha phosphorylation and inhibited protein synthesis in CHO-K1 cells, indicating that the effectiveness of endogenous ceramide could be limited by access to signaling pathways. Thus, expansion of the ER ceramide pool by D609 was not implicated in early (eIF2alpha phosphorylation) or late (apoptotic) cytotoxic events.

Oxysterol binding protein (OSBP) translocation between Golgi and vesicular/cytoplasmic compartments is affected by conditions that alter cholesterol and sphingomyelin homeostasis, indicating a role in lipid and sterol regulation in this organelle. In this study, we show that OSBP dissociation from the Golgi apparatus was inhibited when LDL cholesterol efflux from lysosomes was blocked in Niemann-Pick C (NPC) or U18666A [3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one]-treated fibroblasts. Dissociation of OSBP from the Golgi apparatus in response to LDL was independent of de novo cholesterol biosynthesis. OSBP did not localize with filipin-stained lysosomal cholesterol, and the NPC defect did not alter OSBP expression or phosphorylation. However, OSBP in the Golgi apparatus was progressively dephosphorylated (as assessed by a molecular mass shift on SDS-PAGE) in U18666A-treated fibroblasts or Chinese hamster ovary cells as a result of combined inhibition of LDL cholesterol transport and de novo cholesterol synthesis. In vivo phosphopeptide mapping and mutagenesis of OSBP was used to identify the cholesterol-sensitive phosphorylation sites at serines 381, 384, and 387 that were responsible for the altered mobility on SDS-PAGE. NPC-1 protein-mediated release of LDL-derived cholesterol and de novo biosynthesis regulates OSBP localization and phosphorylation. This indicates that OSBP responds to or senses altered cellular sterol content and transport.

The hallmark of the atherosclerotic lesion is the overaccumulation of cholesterol in arterial wall cells. As no pathway exists for the degradation of cholesterol in peripheral cells, a mechanism is necessary to prevent its accumulation to toxic levels in these cells and to allow its delivery to the liver for excretion in bile. Promoting this reverse cholesterol transport pathway is believed to be the main cardioprotective action of high density lipoprotein (HDL). The rate-limiting step in this pathway is likely the initial removal of cholesterol from peripheral cells by HDL. The pathway HDL utilizes for inducing efflux of excess cellular cholesterol represents an important and as-yet untapped mechanism to employ for the treatment and prevention of atherosclerotic vascular disease. This review summarizes the potential cardioprotective actions of HDL, the mechanisms of HDL-mediated cellular cholesterol efflux, and evidence that the specific pathway of cholesterol removal by HDL may be enhanced and used as a novel target in the therapy of atherosclerosis.

OBJECTIVE:To investigate the effects of positive end-expiratory pressure (PEEP) on end-expiratory lung volume (EELV) and mean oscillated flow rate (VOSC) during high-frequency chest compression (HFCC) in normal subjects and patients with severe COPD.DESIGN:Comparative study.SETTING:Pulmonary function and lung mechanics laboratory, University of Alberta Hospitals.PARTICIPANTS:Six normal subjects (five male; one female) and six patients with clinically stable COPD (five male; one female) with hypercapnia.INTERVENTIONS:A pneumatic vest system was operated at 10 Hz with a mean chest wall pressure of 16 cm H2O to provide the HFCC. A closed-circuit spirometer system permitted measurement of HFCC- and PEEP-induced changes in EELV that were expressed as per cent baseline functional residual capacity (FRC). An isothermic chamber connected near the mouthpiece permitted measurement of VOSC.RESULTS:For the normal subjects, HFCC caused a significant decrease in EELV to 82.0% of FRC (p < or = 0.01) and the addition of 4.8+/-0.5 cm H2O of PEEP during HFCC increased EELV to 97.5% FRC. In the COPD patients, HFCC decreased EELV to 92.3% of FRC (p < or = 0.01), and the addition of 3.7+/-1.0 cm H2O of PEEP increased EELV to 98.4% FRC. For the normal subjects, increasing EELV to near FRC caused VOSC during expiration to increase 14.6% (p < or = 0.01), but there was no significant effect on VOSC during inspiration (5.1% increase). In the COPD patients, PEEP increased VOSC during both inspiration (30.5%) and expiration (57.0%) (both, p < or = 0.01).CONCLUSIONS:Addition of a modest amount of PEEP during HFCC prevents the decrease in EELV and increases VOSC during both phases of spontaneous breathing in COPD patients. This higher VOSC during HFCC+PEEP may improve the effectiveness of HFCC in clearing mucus from the lungs of patients with airway disease.