Exercise-induced changes in myocardial glucose utilization during periods of active cardiac growth.

Exercise training can promote physiological cardiac growth, which has been suggested to involve changes in glucose metabolism to facilitate hypertrophy of cardiomyocytes. In this study, we used a dietary, in vivo isotope labeling approach to examine how exercise training influences the metabolic fate of carbon derived from dietary glucose in the heart during acute, active, and established phases of exercise-induced cardiac growth. Male and female FVB/NJ mice were subjected to treadmill running for up to 4 weeks and cardiac growth was assessed by gravimetry. Cardiac metabolic responses to exercise were assessed via in vivo tracing of [13C6]-glucose via mass spectrometry and nuclear magnetic resonance. We found that the half-maximal cardiac growth response was achieved by approximately 1 week of daily exercise training, with near maximal growth observed in male mice with 2 weeks of training; however, female mice were recalcitrant to exercise-induced cardiac growth and required a higher daily intensity of exercise training to achieve significant, albeit modest, increases in cardiac mass. We also found that increases in the energy charge of adenylate and guanylate nucleotide pools precede exercise-induced changes in cardiac size and were associated with higher glucose tracer enrichment in the TCA pool and in amino acids (aspartate, glutamate) sourced by TCA intermediates. Our data also indicate that the activity of collateral biosynthetic pathways of glucose metabolism may not be markedly altered by exercise. Overall, this study provides evidence that metabolic remodeling in the form of heightened energy charge and increased TCA cycle activity and cataplerosis precedes cardiac growth caused by exercise training in male mice.

Cardiac mitochondrial metabolism during pregnancy and the postpartum period.

The goal of the present study was to characterize changes in mitochondrial respiration in the maternal heart during pregnancy and after birth. Timed pregnancy studies were performed in 12-wk-old female FVB/NJ mice, and cardiac mitochondria were isolated from the following groups of mice: nonpregnant (NP), midpregnancy (MP), late pregnancy (LP), and 1-wk postbirth (PB). Similar to our previous studies, we observed increased heart size during all stages of pregnancy (e.g., MP and LP) and postbirth (e.g., PB) compared with NP mice. Differential cardiac gene and protein expression analyses revealed changes in several mitochondrial transcripts at LP and PB, including several mitochondrial complex subunits and members of the Slc family, important for mitochondrial substrate transport. Respirometry revealed that pyruvate- and glutamate-supported state 3 respiration was significantly higher in PB vs. LP mitochondria, with respiratory control ratio (RCR) values higher in PB mitochondria. In addition, we found that PB mitochondria respired more avidly when given 3-hydroxybutyrate (3-OHB) than mitochondria from NP, MP, and LP hearts, with no differences in RCR. These increases in respiration in PB hearts occurred independent of changes in mitochondrial yield but were associated with higher abundance of 3-hydroxybutyrate dehydrogenase 1. Collectively, these findings suggest that, after birth, maternal cardiac mitochondria have an increased capacity to use 3-OHB, pyruvate, and glutamate as energy sources; however, increases in mitochondrial efficiency in the postpartum heart appear limited to carbohydrate and amino acid metabolism.NEW & NOTEWORTHY Few studies have detailed the physiological adaptations that occur in the maternal heart. We and others have shown that pregnancy-induced cardiac growth is associated with significant changes in cardiac metabolism. Here, we examined mitochondrial respiration and substrate preference in isolated mitochondria from the maternal heart. We show that following birth, cardiac mitochondria are "primed" to respire on carbohydrate, amino acid, and ketone bodies. However, heightened respiratory efficiency is observed only with carbohydrate and amino acid sources. These results suggest that significant changes in mitochondrial respiration occur in the maternal heart in the postpartum period.

Guidelines for in vivo models of developmental programming of cardiovascular disease risk.

Research using animals depends on the generation of offspring for use in experiments or for the maintenance of animal colonies. Although not considered by all, several different factors preceding and during pregnancy, as well as during lactation, can program various characteristics in the offspring. Here, we present the most common models of developmental programming of cardiovascular outcomes, important considerations for study design, and provide guidelines for producing and reporting rigorous and reproducible cardiovascular studies in offspring exposed to normal conditions or developmental insult. These guidelines provide considerations for the selection of the appropriate animal model and factors that should be reported to increase rigor and reproducibility while ensuring transparent reporting of methods and results.

Guidelines for assessing maternal cardiovascular physiology during pregnancy and postpartum.

Maternal mortality rates are at an all-time high across the world and are set to increase in subsequent years. Cardiovascular disease is the leading cause of death during pregnancy and postpartum, especially in the United States. Therefore, understanding the physiological changes in the cardiovascular system during normal pregnancy is necessary to understand disease-related pathology. Significant systemic and cardiovascular physiological changes occur during pregnancy that are essential for supporting the maternal-fetal dyad. The physiological impact of pregnancy on the cardiovascular system has been examined in both experimental animal models and in humans. However, there is a continued need in this field of study to provide increased rigor and reproducibility. Therefore, these guidelines aim to provide information regarding best practices and recommendations to accurately and rigorously measure cardiovascular physiology during normal and cardiovascular disease-complicated pregnancies in human and animal models.

Recommendations for data collection in cohort studies of preterm born individuals - The RECAP Preterm Core Dataset.

BACKGROUND: Preterm birth (before 37 completed weeks of gestation) is associated with an increased risk of adverse health and developmental outcomes relative to birth at term. Existing guidelines for data collection in cohort studies of individuals born preterm are either limited in scope, have not been developed using formal consensus methodology, or did not involve a range of stakeholders in their development. Recommendations meeting these criteria would facilitate data pooling and harmonisation across studies. OBJECTIVES: To develop a Core Dataset for use in longitudinal cohort studies of individuals born preterm. METHODS: This work was carried out as part of the RECAP Preterm project. A systematic review of variables included in existing core outcome sets was combined with a scoping exercise conducted with experts on preterm birth. The results were used to generate a draft core dataset. A modified Delphi process was implemented using two stages with three rounds each. Three stakeholder groups participated: RECAP Preterm project partners; external experts in the field; people with lived experience of preterm birth. The Delphi used a 9-point Likert scale. Higher values indicated greater importance for inclusion. Participants also suggested additional variables they considered important for inclusion which were voted on in later rounds. RESULTS: An initial list of 140 data items was generated. Ninety-six participants across 22 countries participated in the Delphi, of which 29% were individuals with lived experience of preterm birth. Consensus was reached on 160 data items covering Antenatal and Birth Information, Neonatal Care, Mortality, Administrative Information, Organisational Level Information, Socio-economic and Demographic information, Physical Health, Education and Learning, Neurodevelopmental Outcomes, Social, Lifestyle and Leisure, Healthcare Utilisation and Quality of Life. CONCLUSIONS: This core dataset includes 160 data items covering antenatal care through outcomes in adulthood. Its use will guide data collection in new studies and facilitate pooling and harmonisation of existing data internationally.

Rodent Models of Alzheimer's Disease: Past Misconceptions and Future Prospects.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with no effective treatments, not least due to the lack of authentic animal models. Typically, rodent models recapitulate the effects but not causes of AD, such as cholinergic neuron loss: lesioning of cholinergic neurons mimics the cognitive decline reminiscent of AD but not its neuropathology. Alternative models rely on the overexpression of genes associated with familial AD, such as amyloid precursor protein, or have genetically amplified expression of mutant tau. Yet transgenic rodent models poorly replicate the neuropathogenesis and protein overexpression patterns of sporadic AD. Seeding rodents with amyloid or tau facilitates the formation of these pathologies but cannot account for their initial accumulation. Intracerebral infusion of proinflammatory agents offer an alternative model, but these fail to replicate the cause of AD. A novel model is therefore needed, perhaps similar to those used for Parkinson's disease, namely adult wildtype rodents with neuron-specific (dopaminergic) lesions within the same vulnerable brainstem nuclei, 'the isodendritic core', which are the first to degenerate in AD. Site-selective targeting of these nuclei in adult rodents may recapitulate the initial neurodegenerative processes in AD to faithfully mimic its pathogenesis and progression, ultimately leading to presymptomatic biomarkers and preventative therapies.

Cell wall organic matrix composition and biomineralization across reef-building coralline algae under global change.

Crustose coralline algae (CCA) are one of the most important benthic substrate consolidators on coral reefs through their ability to deposit calcium carbonate on an organic matrix in their cell walls. Discrete polysaccharides have been recognized for their role in biomineralization, yet little is known about the carbohydrate composition of organic matrices across CCA taxa and whether they have the capacity to modulate their organic matrix constituents amidst environmental change, particularly the threats of ocean acidification (OA) and warming. We simulated elevated pCO2 and temperature (IPCC RCP 8.5) and subjected four mid-shelf Great Barrier Reef species of CCA to 2 months of experimentation. To assess the variability in surficial monosaccharide composition and biomineralization across species and treatments, we determined the monosaccharide composition of the polysaccharides present in the cell walls of surficial algal tissue and quantified calcification. Our results revealed dissimilarity among species' monosaccharide constituents, which suggests that organic matrices are composed of different polysaccharides across CCA taxa. We also observed that species differentially modulate composition in response to ocean acidification and warming. Our findings suggest that both variability in composition and ability to modulate monosaccharide abundance may play a crucial role in surficial biomineralization dynamics under the stress of OA and global warming.

Coordinated Metabolic Responses Facilitate Cardiac Growth in Pregnancy and Exercise.

PURPOSE OF REVIEW: Pregnancy and exercise are systemic stressors that promote physiological growth of the heart in response to repetitive volume overload and maintenance of cardiac output. This type of remodeling is distinct from pathological hypertrophy and involves different metabolic mechanisms that facilitate growth; however, it remains unclear how metabolic changes in the heart facilitate growth and if these processes are similar in both pregnancy- and exercise-induced cardiac growth. RECENT FINDINGS: The ability of the heart to metabolize a myriad of substrates balances cardiac demands for energy provision and anabolism. During pregnancy, coordination of hormonal status with cardiac reductions in glucose oxidation appears important for physiological growth. During exercise, a reduction in cardiac glucose oxidation also appears important for physiological growth, which could facilitate shuttling of glucose-derived carbons into biosynthetic pathways for growth. Understanding the metabolic underpinnings of physiological cardiac growth could provide insight to optimize cardiovascular health and prevent deleterious remodeling, such as that which occurs from postpartum cardiomyopathy and heart failure. This short review highlights the metabolic mechanisms known to facilitate pregnancy-induced and exercise-induced cardiac growth, both of which require changes in cardiac glucose metabolism for the promotion of growth. In addition, we mention important similarities and differences of physiological cardiac growth in these models as well as discuss current limitations in our understanding of metabolic changes that facilitate growth.

Bemarituzumab in patients with FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma (FIGHT): a randomised, double-blind, placebo-controlled, phase 2 study.

BACKGROUND: Outcomes are poor in patients with HER2-negative, advanced gastric or gastro-oesophageal junction adenocarcinomas. In this study, we investigated efficacy and safety of the first-in-class, afucosylated, humanised IgG1 anti-fibroblast growth factor receptor 2 isoform IIb (FGFR2b) monoclonal antibody bemarituzumab with modified 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) in patients with FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma. METHODS: In the randomised, double-blind, placebo-controlled phase 2 trial (FIGHT), patients aged 18 years and older with HER2 non-positive, FGFR2b-selected gastric or gastro-oesophageal junction adenocarcinoma, and an Eastern Cooperative Oncology Group performance status of 0-1 were recruited from 144 clinical sites across 17 countries. Patients with previous treatment with any selective inhibitor of the FGF-FGFR pathway were excluded. Eligible patients were randomly assigned (1:1), using permuted-block randomisation (block size of four) and a central interactive voice-web-based response system, stratified by geographical region, previous treatment with curative intent, and administration of mFOLFOX6 while being screened for FGFR2b status, to either bemarituzumab (15 mg/kg of bodyweight) or matched placebo intravenously every 2 weeks. All patients also received mFOLFOX6 (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, and 5-fluorouracil as a 400 mg/m2 bolus followed by 2400 mg/m2 over approximately 46 h) intravenously every 2 weeks. Patients were given treatment until disease progression (defined by Response Evaluation Criteria in Solid Tumours [RECIST] version 1.1), unacceptable toxicity, withdrawal of consent, or death. The primary endpoint was progression-free survival in the intention-to-treat population (defined as all patients randomly assigned to treatment). Safety was assessed in all patients who received at least one dose of assigned treatment. This study is registered with ClinicalTrials.gov, NCT03694522, and is now complete. FINDINGS: Between Nov 14, 2017, and May 8, 2020, 910 patients were screened and 155 were randomly assigned to the bemarituzumab (n=77) or placebo group (n=78). Median age was 60·0 years (IQR 51·0-67·0), 44 (28%) participants were women, 111 (72%) were men, 89 (57%) were Asian, and 61 (39%) were White. At the time of the primary analysis and at a median follow-up of 10·9 months (IQR 6·3-14·2), median progression-free survival was 9·5 months (95% CI 7·3-12·9) in the bemarituzumab group and 7·4 months (5·8-8·4) in the placebo group (hazard ratio [HR] 0·68 [95% CI 0·44-1·04; p=0·073). Common grade 3 or worse adverse events were decreased neutrophil count (23 [30%] of 76 in the bemarituzumab group vs 27 [35%] of 77 in the placebo group), cornea disorder (18 [24%] vs none), neutropenia (ten [13%] vs seven [9%]), stomatitis (seven [9%] vs one [1%]), and anaemia (six [8%] vs ten [13%]). Serious treatment-emergent adverse events were reported in 24 (32%) patients in the bemarituzumab group and 28 (36%) in the placebo group. Serious mFOLFOX6 treatment-related adverse events occurred in nine (12%) patients in the bemarituzumab group and in 15 (19%) patients in the placebo group. All-grade corneal events (adverse events of special interest) occurred in 51 (67%) patients in the bemarituzumab group and eight (10%) in the placebo group; grade 3 corneal events were reported only in 18 (24%) patients in the bemarituzumab group. Treatment-related deaths occurred in three patients in the bemarituzumab group (two due to sepsis, one due to pneumonia) and none in the placebo group. INTERPRETATION: In this exploratory phase 2 study, despite no statistically significant improvement in progression-free survival, treatment with bemarituzumab showed promising clinical efficacy. Confirmatory phase 3 trials of bemarituzumab plus mFOLFOX6 powered to demonstrate statistical significance are being investigated in patients with previously untreated, FGFR2b-overexpressing, advanced gastric or gastro-oesophageal junction adenocarcinoma. FUNDING: Five Prime Therapeutics.

Metabolic signatures of pregnancy-induced cardiac growth.

The goal of this study was to develop an atlas of the metabolic, transcriptional, and proteomic changes that occur with pregnancy in the maternal heart. Timed pregnancy studies in FVB/NJ mice revealed a significant increase in heart size by day 8 of pregnancy (midpregnancy; MP), which was sustained throughout the rest of the term compared with nonpregnant control mice. Cardiac hypertrophy and myocyte cross-sectional area were highest 7 days after birth (postbirth; PB) and were associated with significant increases in end-diastolic and end-systolic left ventricular volumes and higher cardiac output. Metabolomics analyses revealed that by day 16 of pregnancy (late pregnancy; LP) metabolites associated with nitric oxide production as well as acylcholines, sphingomyelins, and fatty acid species were elevated, which coincided with a lower activation state of phosphofructokinase and higher levels of pyruvate dehydrogenase kinase 4 (Pdk4) and ß-hydroxybutyrate dehydrogenase 1 (Bdh1). In the postpartum period, urea cycle metabolites, polyamines, and phospholipid levels were markedly elevated in the maternal heart. Cardiac transcriptomics in LP revealed significant increases in not only Pdk4 and Bdh1 but also genes that regulate glutamate and ketone body oxidation, which were preceded in MP by higher expression of transcripts controlling cell proliferation and angiogenesis. Proteomics analysis of the maternal heart in LP and PB revealed significant reductions in several contractile filament and mitochondrial subunit complex proteins. Collectively, these findings describe the coordinated molecular changes that occur in the maternal heart during and after pregnancy.NEW & NOTEWORTHY Little is known of the underlying molecular and cellular mechanisms that contribute to pregnancy-induced cardiac growth. Several lines of evidence suggest that changes in cardiac metabolism may contribute. Here, we provide a comprehensive metabolic atlas of the metabolomic, proteomic, and transcriptomic changes occurring in the maternal heart. We show that pregnancy-induced cardiac growth is associated with changes in glycerophospholipid, nucleotide, and amino acid metabolism, with reductions in cardiac glucose catabolism. Collectively, these results suggest that substantial metabolic changes occur in the maternal heart during and after pregnancy.

Development of a data classification system for preterm birth cohort studies: the RECAP Preterm project.

BACKGROUND: The small sample sizes available within many very preterm (VPT) longitudinal birth cohort studies mean that it is often necessary to combine and harmonise data from individual studies to increase statistical power, especially for studying rare outcomes. Curating and mapping data is a vital first step in the process of data harmonisation. To facilitate data mapping and harmonisation across VPT birth cohort studies, we developed a custom classification system as part of the Research on European Children and Adults born Preterm (RECAP Preterm) project in order to increase the scope and generalisability of research and the evaluation of outcomes across the lifespan for individuals born VPT. METHODS: The multidisciplinary consortium of expert clinicians and researchers who made up the RECAP Preterm project participated in a four-phase consultation process via email questionnaire to develop a topic-specific classification system. Descriptive analyses were calculated after each questionnaire round to provide pre- and post- ratings to assess levels of agreement with the classification system as it developed. Amendments and refinements were made to the classification system after each round. RESULTS: Expert input from 23 clinicians and researchers from the RECAP Preterm project aided development of the classification system's topic content, refining it from 10 modules, 48 themes and 197 domains to 14 modules, 93 themes and 345 domains. Supplementary classifications for target, source, mode and instrument were also developed to capture additional variable-level information. Over 22,000 individual data variables relating to VPT birth outcomes have been mapped to the classification system to date to facilitate data harmonisation. This will continue to increase as retrospective data items are mapped and harmonised variables are created. CONCLUSIONS: This bespoke preterm birth classification system is a fundamental component of the RECAP Preterm project's web-based interactive platform. It is freely available for use worldwide by those interested in research into the long term impact of VPT birth. It can also be used to inform the development of future cohort studies.

The impact of resistance training on strength and correlates of physical activity in youth.

Resistance training (RT) may have a positive impact on specific correlates of physical activity (PA) in inactive and/or obese youth, with strength as a possible underlying mechanism. The aim of this study was to investigate this. Twelve participants (aged 8.9 ± 1.0 years) were assigned to an experimental group (EG) or control group (CG). Pre and post intervention assessments for strength, physical self-perceptions (PSPs), weight status, fundamental movement skills (FMS), and PA levels were completed. The EG participated in a twice-weekly 10-week RT programme. There were significant group x time interactions for FMS (CAMSA total P = 0.016, CAMSA skill score P = 0.036) and stretch stature (P = 0.002) (EG larges changes than the CG). Large effect sizes for the differences in change scores between the EG and CG were evident for CAMSA total score (Hedges' g = 0.830, P = 0.138), CAMSA skill score (Hedges' g = 0.895, P = 0.112) and relative strength (Hedges' g = 0.825, P = 0.140). This study demonstrated that a 10-week RT intervention has a positive effect on strength and FMS, and may also benefit weight status and PSPs. This study supports the development of RT interventions to develop these correlates, and increase PA levels.

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development.

In cereal grain, sucrose is converted into storage carbohydrates: mainly starch, fructan, and mixed-linkage (1,3;1,4)-ß-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley was shown to result in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm, and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose, and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETCs) in transgenic grain were thinner and showed reduced mannan labelling relative to the wild type. At the early storage phase, ruptures of the non-uniformly developed ETCs and disorganization of adjacent endosperm cells were observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.

Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain.

Mobilization of reserves in germinated cereal grains is critical for early seedling vigour, global crop productivity, and hence food security. Gibberellins (GAs) are central to this process. We have developed a spatio-temporal model that describes the multifaceted mechanisms of GA regulation in germinated barley grain. The model was generated using RNA sequencing transcript data from tissues dissected from intact, germinated grain, which closely match measurements of GA hormones and their metabolites in those tissues. The data show that successful grain germination is underpinned by high concentrations of GA precursors in ungerminated grain, the use of independent metabolic pathways for the synthesis of several bioactive GAs during germination, and a capacity to abort bioactive GA biosynthesis. The most abundant bioactive form is GA1, which is synthesized in the scutellum as a glycosyl conjugate that diffuses to the aleurone, where it stimulates de novo synthesis of a GA3 conjugate and GA4. Synthesis of bioactive GAs in the aleurone provides a mechanism that ensures the hormonal signal is relayed from the scutellum to the distal tip of the grain. The transcript data set of 33 421 genes used to define GA metabolism is available as a resource to analyse other physiological processes in germinated grain.

Acute increases in O-GlcNAc indirectly impair mitochondrial bioenergetics through dysregulation of LonP1-mediated mitochondrial protein complex turnover.

The attachment of O-linked ß-N-acetylglucosamine (O-GlcNAc) to the serine and threonine residues of proteins in distinct cellular compartments is increasingly recognized as an important mechanism regulating cellular function. Importantly, the O-GlcNAc modification of mitochondrial proteins has been identified as a potential mechanism to modulate metabolism under stress with both potentially beneficial and detrimental effects. This suggests that temporal and dose-dependent changes in O-GlcNAcylation may have different effects on mitochondrial function. In the current study, we found that acutely augmenting O-GlcNAc levels by inhibiting O-GlcNAcase with Thiamet-G for up to 6 h resulted in a time-dependent decrease in cellular bioenergetics and decreased mitochondrial complex I, II, and IV activities. Under these conditions, mitochondrial number was unchanged, whereas an increase in the protein levels of the subunits of several electron transport complex proteins was observed. However, the observed bioenergetic changes appeared not to be due to direct increased O-GlcNAc modification of complex subunit proteins. Increases in O-GlcNAc were also associated with an accumulation of mitochondrial ubiquitinated proteins; phosphatase and tensin homolog induced kinase 1 (PINK1) and p62 protein levels were also significantly increased. Interestingly, the increase in O-GlcNAc levels was associated with a decrease in the protein levels of the mitochondrial Lon protease homolog 1 (LonP1), which is known to target complex IV subunits and PINK1, in addition to other mitochondrial proteins. These data suggest that impaired bioenergetics associated with short-term increases in O-GlcNAc levels could be due to impaired, LonP1-dependent, mitochondrial complex protein turnover.

Novel role of the ER/SR Ca2+ sensor STIM1 in the regulation of cardiac metabolism.

The endoplasmic reticulum/sarcoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+ entry, is expressed in cardiomyocytes and has been implicated in regulating multiple cardiac processes, including hypertrophic signaling. Interestingly, cardiomyocyte-restricted deletion of STIM1 (crSTIM1-KO) results in age-dependent endoplasmic reticulum stress, altered mitochondrial morphology, and dilated cardiomyopathy in mice. Here, we tested the hypothesis that STIM1 deficiency may also impact cardiac metabolism. Hearts isolated from 20-wk-old crSTIM1-KO mice exhibited a significant reduction in both oxidative and nonoxidative glucose utilization. Consistent with the reduction in glucose utilization, expression of glucose transporter 4 and AMP-activated protein kinase phosphorylation were all reduced, whereas pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphorylation were increased, in crSTIM1-KO hearts. Despite similar rates of fatty acid oxidation in control and crSTIM1-KO hearts ex vivo, crSTIM1-KO hearts contained increased lipid/triglyceride content as well as increased fatty acid-binding protein 4, fatty acid synthase, acyl-CoA thioesterase 1, hormone-sensitive lipase, and adipose triglyceride lipase expression compared with control hearts, suggestive of a possible imbalance between fatty acid uptake and oxidation. Insulin-mediated alterations in AKT phosphorylation were observed in crSTIM1-KO hearts, consistent with cardiac insulin resistance. Interestingly, we observed abnormal mitochondria and increased lipid accumulation in 12-wk crSTIM1-KO hearts, suggesting that these changes may initiate the subsequent metabolic dysfunction. These results demonstrate, for the first time, that cardiomyocyte STIM1 may play a key role in regulating cardiac metabolism. NEW & NOTEWORTHY Little is known of the physiological role of stromal interaction molecule 1 (STIM1) in the heart. Here, we demonstrate, for the first time, that hearts lacking cardiomyocyte STIM1 exhibit dysregulation of both cardiac glucose and lipid metabolism. Consequently, these results suggest a potentially novel role for STIM1 in regulating cardiac metabolism.

Revised Phylogeny of the Cellulose Synthase Gene Superfamily: Insights into Cell Wall Evolution.

Cell walls are crucial for the integrity and function of all land plants and are of central importance in human health, livestock production, and as a source of renewable bioenergy. Many enzymes that mediate the biosynthesis of cell wall polysaccharides are encoded by members of the large cellulose synthase (CesA) gene superfamily. Here, we analyzed 29 sequenced genomes and 17 transcriptomes to revise the phylogeny of the CesA gene superfamily in angiosperms. Our results identify ancestral gene clusters that predate the monocot-eudicot divergence and reveal several novel evolutionary observations, including the expansion of the Poaceae-specific cellulose synthase-like CslF family to the graminids and restiids and the characterization of a previously unreported eudicot lineage, CslM, that forms a reciprocally monophyletic eudicot-monocot grouping with the CslJ clade. The CslM lineage is widely distributed in eudicots, and the CslJ clade, which was thought previously to be restricted to the Poales, is widely distributed in monocots. Our analyses show that some members of the CslJ lineage, but not the newly identified CslM genes, are capable of directing (1,3;1,4)-ß-glucan biosynthesis, which, contrary to current dogma, is not restricted to Poaceae.

Isolation of tissues and preservation of RNA from intact, germinated barley grain.

Isolated barley (Hordeum vulgare L.) aleurone layers have been widely used as a model system for studying gene expression and hormonal regulation in germinating cereal grains. A serious technological limitation of this approach has been the inability to confidently extrapolate conclusions obtained from isolated tissues back to the whole grain, where the co-location of several living and non-living tissues results in complex tissue-tissue interactions and regulatory pathways coordinated across the multiple tissues. Here we have developed methods for isolating fragments of aleurone, starchy endosperm, embryo, scutellum, pericarp-testa, husk and crushed cell layers from germinated grain. An important step in the procedure involves the rapid fixation of the intact grain to freeze the transcriptional activity of individual tissues while dissection is effected for subsequent transcriptomic analyses. The developmental profiles of 19 611 gene transcripts were precisely defined in the purified tissues and in whole grain during the first 24 h of germination by RNA sequencing. Spatial and temporal patterns of transcription were validated against well-defined data on enzyme activities in both whole grain and isolated tissues. Transcript profiles of genes involved in mitochondrial assembly and function were used to validate the very early stages of germination, while the profiles of genes involved in starch and cell wall mobilisation matched existing data on activities of corresponding enzymes. The data will be broadly applicable for the interrogation of co-expression and differential expression patterns and for the identification of transcription factors that are important in the early stages of grain and seed germination.

Genetic and environmental factors contribute to variation in cell wall composition in mature desi chickpea (Cicer arietinum L.) cotyledons.

Chickpea (Cicer arietinum L.) is an important nutritionally rich legume crop that is consumed worldwide. Prior to cooking, desi chickpea seeds are most often dehulled and cleaved to release the split cotyledons, referred to as dhal. Compositional variation between desi genotypes has a significant impact on nutritional quality and downstream processing, and this has been investigated mainly in terms of starch and protein content. Studies in pulses such as bean and lupin have also implicated cell wall polysaccharides in cooking time variation, but the underlying relationship between desi chickpea cotyledon composition and cooking performance remains unclear. Here, we utilized a variety of chemical and immunohistological assays to examine details of polysaccharide composition, structure, abundance, and location within the desi chickpea cotyledon. Pectic polysaccharides were the most abundant cell wall components, and differences in monosaccharide and glycosidic linkage content suggest both environmental and genetic factors contribute to cotyledon composition. Genotype-specific differences were identified in arabinan structure, pectin methylesterification, and calcium-mediated pectin dimerization. These differences were replicated in distinct field sites and suggest a potentially important role for cell wall polysaccharides and their underlying regulatory machinery in the control of cooking time in chickpea.

Method for hull-less barley transformation and manipulation of grain mixed-linkage beta-glucan.

Hull-less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull-less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull-less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over-express genes encoding synthases for the important dietary fiber component, (1,3;1,4)-ß-glucan (mixed-linkage glucan), primarily present in starchy endosperm cell walls. Over-expression of the HvCslF6 gene, driven by an endosperm-specific promoter, produced lines where mixed-linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub-aleurone cells. This work provides proof-of-concept evidence that mixed-linkage glucan content in hull-less barley grain can be increased by over-expression of the HvCslF6 gene, but also indicates that hull-less cultivars may be more sensitive to attempts to modify cell wall composition.