Mechanisms and significance of tissue-specific MICU regulation of the mitochondrial calcium uniporter complex.

Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.

MicroRNA regulation postbleomycin due to the R213G extracellular superoxide dismutase variant is predicted to suppress inflammatory and immune pathways.

Oxidative stress is a key contributor to the development of dysregulated inflammation in acute lung injury (ALI). A naturally occurring single nucleotide polymorphism in the key extracellular antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), results in an arginine to glycine substitution (R213G) that promotes resolution of inflammation and protection against bleomycin-induced ALI. Previously we found that mice harboring the R213G mutation in EC-SOD exhibit a transcriptomic profile consistent with a striking suppression of inflammatory and immune pathways 7 days postbleomycin. However, the alterations in noncoding regulatory RNAs in wild-type (WT) and R213G EC-SOD lungs have not been examined. Therefore, we used next-generation microRNA (miR) Sequencing of lung tissue to identify dysregulated miRs 7 days after bleomycin in WT and R213G mice. Differential expression analysis identified 92 WT and 235 R213G miRs uniquely dysregulated in their respective genotypes. Subsequent pathway analysis identified that these miRs were predicted to regulate approximately half of the differentially expressed genes previously identified. The gene targets of these altered miRs indicate suppression of immune and inflammatory pathways in the R213G mice versus activation of these pathways in WT mice. Triggering receptor expressed on myeloid cells 1 (TREM1) signaling was identified as the inflammatory pathway with the most striking difference between WT and R213G lungs. miR-486b-3p was identified as the most dysregulated miR predicted to regulate the TREM1 pathway. We validated the increase in TREM1 signaling using miR-486b-3p antagomir transfection. These findings indicate that differential miR regulation is predicted to regulate the inflammatory gene profile, contributing to the protection against ALI in R213G mice.

Heart failure in single right ventricle congenital heart disease: physiological and molecular considerations.

Because of remarkable surgical and medical advances over the past several decades, there are growing numbers of infants and children living with single ventricle congenital heart disease (SV), where there is only one functional cardiac pumping chamber. Nevertheless, cardiac dysfunction (and ultimately heart failure) is a common complication in the SV population, and pharmacological heart failure therapies have largely been ineffective in mitigating the need for heart transplantation. Given that there are several inherent risk factors for ventricular dysfunction in the setting of SV in addition to probable differences in molecular adaptations to heart failure between children and adults, it is perhaps not surprising that extrapolated adult heart failure medications have had limited benefit in children with SV heart failure. Further investigations into the molecular mechanisms involved in pediatric SV heart failure may assist with risk stratification as well as development of targeted, efficacious therapies specific to this patient population. In this review, we present a brief overview of SV anatomy and physiology, with a focus on patients with a single morphological right ventricle requiring staged surgical palliation. Additionally, we discuss outcomes in the current era, risk factors associated with the progression to heart failure, present state of knowledge regarding molecular alterations in end-stage SV heart failure, and current therapeutic interventions. Potential avenues for improving SV outcomes, including identification of biomarkers of heart failure progression, implications of personalized medicine and stem cell-derived therapies, and applications of novel models of SV disease, are proposed as future directions.

Alteration of cardiolipin biosynthesis and remodeling in single right ventricle congenital heart disease.

Despite advances in both medical and surgical therapies, individuals with single ventricle heart disease (SV) remain at high risk for the development of heart failure (HF). However, the molecular mechanisms underlying remodeling and eventual HF in patients with SV are poorly characterized. Cardiolipin (CL), an inner mitochondrial membrane phospholipid, is critical for proper mitochondrial function, and abnormalities in CL content and composition are known in various cardiovascular disease etiologies. The purpose of this study was to investigate myocardial CL content and composition in failing and nonfailing single right ventricle (RV) samples compared with normal control RV samples, to assess mRNA expression of CL biosynthetic and remodeling enzymes, and to quantitate relative mitochondrial copy number. A cross-sectional analysis of RV myocardial tissue from 22 failing SV (SVHF), 9 nonfailing SV (SVNF), and 10 biventricular control samples (BVNF) was performed. Expression of enzymes involved in CL biosynthesis and remodeling were analyzed using RT-qPCR and relative mitochondrial DNA copy number determined by qPCR. Normal phase high-pressure liquid chromatography coupled to electrospray ionization mass spectrometry was used to quantitate total and specific CL species. While mitochondrial copy number was not significantly different between groups, total CL content was significantly lower in SVHF myocardium compared with BVNF controls. Despite having lower total CL content however, the relative percentage of the major tetralinoleoyl CL species is preserved in SVHF samples relative to BVNF controls. Correspondingly, expression of enzymes involved in CL biosynthesis and remodeling were upregulated in SVHF samples when compared with both SVNF samples and BVNF controls.NEW & NOTEWORTHY The mechanisms underlying heart failure in the single ventricle (SV) congenital heart disease population are largely unknown. In this study we identify alterations in cardiac cardiolipin metabolism, composition, and content in children with SV heart disease. These findings suggest that cardiolipin could be a novel therapeutic target in this unique population of patients.

Important Considerations in Pediatric Heart Failure.

PURPOSE OF REVIEW: The goal of this paper is to provide an overview of contemporary knowledge specific to the causes, management, and outcome of heart failure in children. RECENT FINDINGS: While recently there have been subtle improvements in heart failure outcomes in children, these improvements lag significantly behind that of adults. There is a growing body of literature suggesting that pediatric heart failure is a unique disease process with age- and disease-specific myocardial adaptations. In addition, the heterogenous etiologies of heart failure in children contribute to differential response to therapies and challenge the ability to obtain meaningful results from prospective clinical trials. Consideration of novel clinical trial designs with achievable but clinically relevant endpoints and focused study of the mechanisms underlying pediatric heart failure secondary to cardiomyopathies and structural heart disease are essential if we hope to advance care and identify targeted and effective therapies.

Probenecid Improves Cardiac Function in Subjects with a Fontan Circulation and Augments Cardiomyocyte Calcium Homeostasis.

Subjects with functionally univentricular circulation who have completed staged single ventricle palliation, with the final stage culminating in the Fontan procedure, are often living into adulthood. However, high morbidity and mortality remain prevalent in these patients, as diastolic and systolic dysfunction of the single systemic ventricle are linked to Fontan circulatory failure. We presently investigated the effects of probenecid in post-Fontan patients. Used for decades for the treatment of gout, probenecid has been shown in recent years to positively influence cardiac function via effects on the Transient Receptor Potential Vanilloid 2 (TRPV2) channel in cardiomyocytes. Indeed, we observed that probenecid improved cardiac function and exercise performance in patients with a functionally univentricular circulation. This was consistent with our findings from a retrospective cohort of patients with single ventricle physiology where TRPV2 expression was increased. Experiments in isolated cardiomyocytes associated these positive actions to augmentation of diastolic calcium homeostasis.

Increased myocyte calcium sensitivity in end-stage pediatric dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure (HF) in children, resulting in high mortality and need for heart transplantation. The pathophysiology underlying pediatric DCM is largely unclear; however, there is emerging evidence that molecular adaptations and response to conventional HF medications differ between children and adults. To gain insight into alterations leading to systolic dysfunction in pediatric DCM, we measured cardiomyocyte contractile properties and sarcomeric protein phosphorylation in explanted pediatric DCM myocardium (N = 8 subjects) compared with nonfailing (NF) pediatric hearts (N = 8 subjects). Force-pCa curves were generated from skinned cardiomyocytes in the presence and absence of protein kinase A. Sarcomeric protein phosphorylation was quantified with Pro-Q Diamond staining after gel electrophoresis. Pediatric DCM cardiomyocytes demonstrate increased calcium sensitivity (pCa50 =5.70 ± 0.0291), with an associated decrease in troponin (Tn)I phosphorylation compared with NF pediatric cardiomyocytes (pCa50 =5.59 ± 0.0271, P = 0.0073). Myosin binding protein C and TnT phosphorylation are also lower in pediatric DCM, whereas desmin phosphorylation is increased. Pediatric DCM cardiomyocytes generate peak tension comparable to that of NF pediatric cardiomyocytes [DCM 29.7 mN/mm2, interquartile range (IQR) 21.5-49.2 vs. NF 32.8 mN/mm2, IQR 21.5-49.2 mN/mm2; P = 0.6125]. In addition, cooperativity is decreased in pediatric DCM compared with pediatric NF (Hill coefficient: DCM 1.56, IQR 1.31-1.94 vs. NF 1.94, IQR 1.36-2.86; P = 0.0425). Alterations in sarcomeric phosphorylation and cardiomyocyte contractile properties may represent an impaired compensatory response, contributing to the detrimental DCM phenotype in children.NEW & NOTEWORTHY Our study is the first to demonstrate that cardiomyocytes from infants and young children with dilated cardiomyopathy (DCM) exhibit increased calcium sensitivity (likely mediated by decreased troponin I phosphorylation) compared with nonfailing pediatric cardiomyocytes. Compared with published values in adult cardiomyocytes, pediatric cardiomyocytes have notably decreased cooperativity, with a further reduction in the setting of DCM. Distinct adaptations in cardiomyocyte contractile properties may contribute to a differential response to pharmacological therapies in the pediatric DCM population.

R213G polymorphism in SOD3 protects against bleomycin-induced inflammation and attenuates induction of proinflammatory pathways.

Extracellular superoxide dismutase (EC-SOD), one of three mammalian SOD isoforms, is the sole extracellular enzymatic defense against superoxide. A known human single nucleotide polymorphism (SNP) in the matrix-binding domain of EC-SOD characterized by an arginine-to-glycine substitution at position 213 (R213G) redistributes EC-SOD from the matrix into extracellular fluids. We previously reported that knock-in mice harboring the human R213G SNP (R213G mice) exhibited enhanced resolution of inflammation with subsequent protection against fibrosis following bleomycin treatment compared with wild-type (WT) littermates. Herein we set out to determine the underlying pathways with RNA-Seq analysis of WT and R213G lungs 7 days post-PBS and bleomycin. RNA-Seq analysis uncovered significant differential gene expression changes induced in WT and R213G strains in response to bleomycin. Ingenuity Pathways Analysis was used to predict differentially regulated up- and downstream processes based on transcriptional changes. Most prominent was the induction of inflammatory and immune responses in WT mice, which were suppressed in the R213G mice. Specifically, PKC signaling in T lymphocytes, IL-6, and NFΚB signaling were opposed in WT mice when compared with R213G. Several upstream regulators such as IFNγ, IRF3, and IKBKG were implicated in the divergent responses between WT and R213G mice. Our data suggest that the redistributed EC-SOD due to the R213G SNP attenuates the dysregulated inflammatory responses observed in WT mice. We speculate that redistributed EC-SOD protects against dysregulated alveolar inflammation via reprogramming of recruited immune cells toward a proresolving state.

Fibrosis-Related Gene Expression in Single Ventricle Heart Disease.

OBJECTIVE: To evaluate fibrosis and fibrosis-related gene expression in the myocardium of pediatric subjects with single ventricle with right ventricular failure. STUDY DESIGN: Real-time quantitative polymerase chain reaction was performed on explanted right ventricular myocardium of pediatric subjects with single ventricle disease and controls with nonfailing heart disease. Subjects were divided into 3 groups: single ventricle failing (right ventricular failure before or after stage I palliation), single ventricle nonfailing (infants listed for primary transplantation with normal right ventricular function), and stage III (Fontan or right ventricular failure after stage III). To evaluate subjects of similar age and right ventricular volume loading, single ventricle disease with failure was compared with single ventricle without failure and stage III was compared with nonfailing right ventricular disease. Histologic fibrosis was assessed in all hearts. Mann-Whitney tests were performed to identify differences in gene expression. RESULTS: Collagen (Col1α, Col3) expression is decreased in single ventricle congenital heart disease with failure compared with nonfailing single ventricle congenital heart disease (P = .019 and P = .035, respectively), and is equivalent in stage III compared with nonfailing right ventricular heart disease. Tissue inhibitors of metalloproteinase (TIMP-1, TIMP-3, and TIMP-4) are downregulated in stage III compared with nonfailing right ventricular heart disease (P = .0047, P = .013 and P = .013, respectively). Matrix metalloproteinases (MMP-2, MMP-9) are similar between nonfailing single ventricular heart disease and failing single ventricular heart disease, and between stage III heart disease and nonfailing right ventricular heart disease. There is no difference in the prevalence of right ventricular fibrosis by histology in subjects with single ventricular failure heart disease with right ventricular failure (18%) compared with those with normal right ventricular function (38%). CONCLUSIONS: Fibrosis is not a primary contributor to right ventricular failure in infants and young children with single ventricular heart disease. Additional studies are required to understand whether antifibrotic therapies are beneficial in this population.

Cardiac Transcriptome Remodeling and Impaired Bioenergetics in Single-Ventricle Congenital Heart Disease.

The mechanisms responsible for heart failure in single-ventricle congenital heart disease are unknown. Using explanted heart tissue, we showed that failing single-ventricle hearts have dysregulated metabolic pathways, impaired mitochondrial function, decreased activity of carnitine palmitoyltransferase activity, and altered functioning of the tricarboxylic acid cycle. Interestingly, nonfailing single-ventricle hearts demonstrated an intermediate metabolic phenotype suggesting that they are vulnerable to development of heart failure in the future. Mitochondrial targeted therapies and treatments aimed at normalizing energy generation could represent a novel approach to the treatment or prevention of heart failure in this vulnerable group of patients.

Medical Therapies for Heart Failure in Hypoplastic Left Heart Syndrome.

Significant surgical and medical advances over the past several decades have resulted in a growing number of infants and children surviving with hypoplastic left heart syndrome (HLHS) and other congenital heart defects associated with a single systemic right ventricle (RV). However, cardiac dysfunction and ultimately heart failure (HF) remain the most common cause of death and indication for transplantation in this population. Moreover, while early recognition and treatment of single ventricle-related complications are essential to improving outcomes, there are no proven therapeutic strategies for single systemic RV HF in the pediatric population. Importantly, prototypical adult HF therapies have been relatively ineffective in mitigating the need for cardiac transplantation in HLHS, likely due to several unique attributes of the failing HLHS myocardium. Here, we discuss the most commonly used medical therapies for the treatment of HF symptoms in HLHS and other single systemic RV patients. Additionally, we provide an overview of potential novel therapies for systemic ventricular failure in the HLHS and related populations based on fundamental science, pre-clinical, clinical, and observational studies in the current literature.

Linoleate-Enrichment of Mitochondrial Cardiolipin Molecular Species Is Developmentally Regulated and a Determinant of Metabolic Phenotype.

Cardiolipin (CL), the major mitochondrial phospholipid, regulates the activity of many mitochondrial membrane proteins. CL composition is shifted in heart failure with decreases in linoleate and increases in oleate side chains, but whether cardiolipin composition directly regulates metabolism is unknown. This study defines cardiolipin composition in rat heart and liver at three distinct ages to determine the influence of CL composition on beta-oxidation (ß-OX). CL species, expression of ß-OX and glycolytic genes, and carnitine palmitoyltransferase (CPT) activity were characterized in heart and liver from neonatal, juvenile, and adult rats. Ventricular myocytes were cultured from neonatal, juvenile, and adult rats and cardiolipin composition and CPT activity were measured. Cardiolipin composition in neonatal rat ventricular cardiomyocytes (NRVMs) was experimentally altered and mitochondrial respiration was assessed. Linoleate-enrichment of CL was observed in rat heart, but not liver, with increasing age. ß-OX genes and CPT activity were generally higher in adult heart and glycolytic genes lower, as a function of age, in contrast to liver. Palmitate oxidation increased in NRVMs when CL was enriched with linoleate. Our results indicate (1) CL is developmentally regulated, (2) linoleate-enrichment is associated with increased ß-OX and a more oxidative mitochondrial phenotype, and (3) experimentally induced linoleate-enriched CL in ventricular myocytes promotes a shift from pyruvate metabolism to fatty acid ß-OX.

Serum circulating proteins from pediatric patients with dilated cardiomyopathy cause pathologic remodeling and cardiomyocyte stiffness.

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remain limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with serum from nonfailing or DCM pediatric patients activates the fetal gene program (FGP). Here we show that serum treatment with proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating miRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is upregulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA-Seq, indicated extracellular matrix remodeling and focal adhesion pathways were upregulated in pediatric DCM serum and in DCM serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled-related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM serum treatment. Our results show that serum circulating proteins promoted pathological changes in gene expression and cellular stiffness, and circulating miRNAs were protective against pathological changes.

Optimization of phenol-chloroform RNA extraction.

Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. •Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.

Phosphodiesterase-5 Is Elevated in Failing Single Ventricle Myocardium and Affects Cardiomyocyte Remodeling In Vitro.

Background Single ventricle (SV) congenital heart disease is fatal without intervention, and eventual heart failure is a major cause of morbidity and mortality. Although there are no proven medical therapies for the treatment or prevention of heart failure in the SV heart disease population, phosphodiesterase-5 inhibitors (PDE5i), such as sildenafil, are increasingly used. Although the pulmonary vasculature is the primary target of PDE5i therapy in patients with SV heart disease, the effects of PDE5i on the SV heart disease myocardium remain largely unknown. We sought to determine PDE5 expression and activity in the single right ventricle of SV heart disease patients relative to nonfailing controls and to determine whether PDE5 impacts cardiomyocyte remodeling using a novel serum-based in vitro model. Methods and Results PDE5 expression (n=9 nonfailing; n=7 SV heart disease), activity (n=8 nonfailing; n=9 SV heart disease), and localization (n=3 SV heart disease) were determined in explanted human right ventricle myocardium. PDE5 is expressed in SV heart disease cardiomyocytes, and PDE5 protein expression and activity are increased in SV heart disease right ventricle compared with nonfailing right ventricle. Isolated neonatal rat ventricular myocytes were treated for 72 hours with nonfailing or SV heart disease patient serum±sildenafil. Reverse transcription quantitative polymerase chain reaction (n=5 nonfailing; n=12 SV heart disease) and RNA sequencing (n=3 nonfailing; n=3 SV heart disease) were performed on serum-treated neonatal rat ventricular myocytes and demonstrated that treatment with SV heart disease sera results in pathological gene expression changes that are attenuated with PDE5i. Conclusions PDE5 is increased in failing SV heart disease myocardium, and pathological gene expression changes in SV heart disease serum-treated neonatal rat ventricular myocytes are abrogated by PDE5i. These results suggest that PDE5 represents an intriguing myocardial therapeutic target in this population.

Glucose or Altered Ceramide Biosynthesis Mediate Oxygen Deprivation Sensitivity Through Novel Pathways Revealed by Transcriptome Analysis in Caenorhabditis elegans.

Individuals with type 2 diabetes display metabolic abnormalities, such as hyperglycemia, increased free fatty acids, insulin resistance, and altered ceramide levels, that contribute to vascular dysfunctions and compromised oxygen delivery. Caenorhabditis elegans fed a glucose-supplemented diet or with altered ceramide metabolism, due to a hyl-2 mutation, are sensitive to oxygen deprivation (anoxia). Our experiments showed that the combination of these factors further decreased the anoxia survival. RNA-sequencing analysis was performed to assess how a glucose-supplemented diet and/or a hyl-2 mutation altered the transcriptome. Comparison analysis of transcripts associated with anoxia-sensitive animals [hyl-2(tm2031) mutation or a glucose diet] revealed 199 common transcripts encoded by genes with known or predicted functions involving innate immunity, cuticle function (collagens), or xenobiotic and endobiotic phase I and II detoxification system. Use of RNA interference (RNAi) to target gene products of the xenobiotic and endobiotic phase I and II detoxification system (UDP-glycosyltransferase and Cytochrome p450 genes; ugt-15, ugt-18, ugt-19, ugt-41, ugt-63, cyp-13A12, cyp-25A1, and cyp-33C8) increased anoxia survival in wild-type animals fed a standard diet. Anoxia sensitivity of the hyl-2(tm2031) animals was suppressed by RNAi of cyp-25A1 or cyp-33C8 genes. A glucose diet fed to the P0 hermaphrodite decreased the anoxia survival of its F1 embryos; however, the RNAi of ugt-63 and cyp-33C8 suppressed anoxia sensitivity. These studies provide evidence that the detoxification system impacts oxygen deprivation responses and that C. elegans can be used to model the conserved detoxification system.

Glucose induces sensitivity to oxygen deprivation and modulates insulin/IGF-1 signaling and lipid biosynthesis in Caenorhabditis elegans.

Diet is a central environmental factor that contributes to the phenotype and physiology of individuals. At the root of many human health issues is the excess of calorie intake relative to calorie expenditure. For example, the increasing amount of dietary sugars in the human diet is contributing to the rise of obesity and type 2 diabetes. Individuals with obesity and type 2 diabetes have compromised oxygen delivery, and thus it is of interest to investigate the impact a high-sugar diet has on oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, we determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide synthesis, modulates anoxia survival. Additionally, a glucose-supplemented diet alters lipid localization and initiates a positive chemotaxis response. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified show homology to human genes that are differentially regulated in response to obesity or type 2 diabetes, suggesting that there may be conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans. These findings support the utility of the C. elegans model for understanding the molecular mechanisms regulating dietary-induced metabolic diseases.

Caenorhabditis elegans: an old genetic model can learn new epigenetic tricks.

Gamete cells pass on information to the next generation via DNA sequence and also through epigenetic mechanisms such as small RNAs, DNA methylation, or chromatin modifications. Caenorhabditis elegans is a genetic model system that an enormous number of talented researchers have used to understand biological phenomenon and develop molecular tools that have ultimately led to paradigm-shifting ideas in biology. Thus, this model is well poised to further investigate the molecular mechanisms involved with epigenetic modifications and transgenerational epigenetic inheritance. The strengths of this model system include a historical wealth of information regarding genetics, development, germline function, chromosome biology, and the regulation of gene expression. Using this system, one can investigate the mechanisms involved with how the germline passes on heritable epigenetic information to subsequent generations. Here, we highlight aspects about the biology of C. elegans that make it amenable to epigenetic studies, highlight some recent findings in the field of epigenetics, and comment on how this system would be beneficial for future biological studies involving epigenetic processes.

Use of time lapse microscopy to visualize anoxia-induced suspended animation in C. elegans embryos.

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins(1-4). In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; <2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.