scholarly journals Activation of amino acid metabolic program in response to impaired glycolysis in cardiac HIF1 deficient mice

2020 ◽  
Author(s):  
Ivan Menendez-Montes ◽  
Beatriz Escobar ◽  
Manuel J. Gomez ◽  
Teresa Albendea-Gomez ◽  
Beatriz Palacios ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heart Development ◽  
Alternative Energy ◽  
Therapeutic Interventions ◽  
Acid Oxidation ◽  
Normal Fatty Acid ◽  
Loss Of Function ◽  
Cardiac Progenitors ◽  
Knock Out ◽  
The Impact

ABSTRACTHypoxia is an important environmental cue in heart development. Despite of extensive characterization of gain and loss of function models, there is disagreement about the impact of HIF1α elimination in cardiac tissue. Here, we used a new conditional knock out of Hif1a in NKX2.5 cardiac progenitors to assess the morphological and functional consequences of HIF1α loss in the developing heart. By combining histology, electron microscopy and high-throughout genomics, proteomics and metabolomics, we found that deletion of Hif1a leads to impaired embryonic glycolysis without influencing cardiomyocyte proliferation and results in an increased mitochondrial number, activation of a transient amino acid response and upregulation of HIF2α and ATF4 by E12.5. Hif1a mutants display normal fatty acid oxidation metabolic profile and do not show any sign of cardiac dysfunction in the adulthood. Our results demonstrate that HIF1 signaling is dispensable for heart development and reveal the metabolic flexibility of the embryonic myocardium, opening the potential application of alternative energy sources as therapeutic interventions during ischemic events.

Abstract 16335: Metabolic Reprogramming From Glycolysis to Amino Acid Utilization in Cardiac Hif1 Alpha Deficient Mice

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ivan Menendez-Montes ◽  
Beatriz Escobar ◽  
Manuel J Gómez ◽  
Teresa Albendea-Gomez ◽  
Beatriz Palacios ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heart Development ◽  
Alternative Fuels ◽  
Cardiac Injury ◽  
Cardiac Metabolism ◽  
Metabolic Reprogramming ◽  
Structural Defects ◽  
Acid Oxidation ◽  
Cardiac Progenitors ◽  
Knock Out

Introduction: Hypoxia is an important environmental cue implicated in several physiopathological processes, including cardiac development. Several gain of function models described before indicate that HIF1 signaling needs to be tightly regulated to ensure proper heart formation. However, there is lack of consensus about the functional outcomes of cardiac HIF1 elimination. We have previously reported that HIF1alpha expression is spatiotemporally regulated along cardiogenesis, establishing metabolic territories in the embryonic myocardium and controlling a switch from glycolysis to fatty acid oxidation (FAO) essential for chamber formation and cardiomyocyte maturation. Objectives and Hypothesis: We aim to assess the consequences of cardiac deletion of HIF1alpha during heart development and identify the adaptations to HIF1 signaling loss. Based on the tight regulation of HIF1alpha expression during cardiogenesis, we anticipated significant alterations of cardiac metabolism as well as functional and structural defects in HIF1alpha mutants. Methods and Results: A new conditional Hif1alpha knock out was generated in NKX2.5 cardiac progenitors. By means of histology, electron microscopy and high-throughput genomics, proteomics and metabolomics, we found that deletion of Hif1alpha leads to impaired embryonic glycolysis without influencing cardiomyocyte size or proliferation and results in increased mitochondrial number, transient activation of amino acid response and upregulation of HIF2alpha and ATF4. HIF1alpha mutants display normal FAO metabolic profile and do not show cardiac dysfunction in the adulthood. Conclusions: We demonstrated that HIF1 signaling is dispensable for heart development and uncovered the metabolic flexibility of the mammalian embryonic myocardium, able to utilize alternative fuels to carbohydrates in contrast to other vertebrates like zebrafish. This data highlights the importance of HIF in cardiac metabolic programing and could explain the distinct proliferative and regenerative capacity of cardiomyocytes from different species in response to cardiac injury.

scholarly journals Metabolic reprogramming from glycolysis to amino acid utilization in cardiac HIF1α deficient mice

2019 ◽  
Author(s):  
Ivan Menendez-Montes ◽  
Beatriz Escobar ◽  
Beatriz Palacios ◽  
Manuel J. Gomez ◽  
Elena Bonzon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heart Development ◽  
Cardiac Development ◽  
Building Blocks ◽  
Embryonic Heart ◽  
Metabolic Reprogramming ◽  
Acid Oxidation ◽  
Metabolic Switch ◽  
Cardiac Growth ◽  
Cardiac Progenitors

AbstractRationaleHypoxia is an important environmental cue implicated in several physiopathological processes, including heart development. Several mouse models of activation or inhibition of hypoxia have been previously described. While gain of function models have been extensively characterized and indicate that HIF1 signaling needs to be tightly regulated to ensure a proper cardiac development, there is lack of consensus in the field about the functional outcomes of HIF1α loss.ObjectiveIn this study, we aim to assess the consequences of cardiac deletion of HIF1α during heart development and identify the cardiac adaptations to HIF1 loss.Methods and ResultsHere, we used a conditional deletion model ofHif1ain NKX2.5+cardiac progenitors. By a combination of histology, electron microscopy, massive gene expression studies, proteomics, metabolomics and cardiac imaging, we found that HIF1α is dispensable for cardiac development.Hif1aloss results in glycolytic inhibition in the embryonic heart without affecting normal cardiac growth. However, together with a premature increase in mitochondrial number by E12.5, we found global upregulation of amino acid transport and catabolic processes. Interestingly, this amino acid catabolism activation is transient and does not preclude the normal cardiac metabolic switch towards fatty acid oxidation (FAO) after E14.5. Moreover,Hif1aloss is accompanied by an increase in ATF4, described as an important regulator of several amino acid transporters.ConclusionsOur data indicate that HIF1α is not required for normal cardiac development and suggest that additional mechanisms can compensateHif1aloss. Moreover, our results reveal the metabolic flexibility of the embryonic heart at early stages of development, showing the capacity of the myocardium to adapt its energy source to satisfy the energetic and building blocks demands to achieve normal cardiac growth and function. This metabolic reprograming might be relevant in the setting of adult cardiac failure.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

scholarly journals SARS-CoV-2 Proteins Exploit Host’s Genetic and Epigenetic Mediators for the Annexation of Key Host Signaling Pathways

2021 ◽  
Vol 7 ◽  
Author(s):  
Md. Abdullah-Al-Kamran Khan ◽  
Abul Bashar Mir Md. Khademul Islam
Keyword(s):  
Signaling Pathways ◽  
Heart Development ◽  
Kidney Development ◽  
Viral Proteins ◽  
Differentially Expressed ◽  
Therapeutic Interventions ◽  
Acid Oxidation ◽  
Immune Signaling ◽  
Antiviral Immune Response ◽  
Host Signaling

The constant rise of the death toll and cases of COVID-19 has made this pandemic a serious threat to human civilization. Understanding of host-SARS-CoV-2 interaction in viral pathogenesis is still in its infancy. In this study, we utilized a blend of computational and knowledgebase approaches to model the putative virus-host interplay in host signaling pathways by integrating the experimentally validated host interactome proteins and differentially expressed host genes in SARS-CoV-2 infection. While searching for the pathways in which viral proteins interact with host proteins, we discovered various antiviral immune response pathways such as hypoxia-inducible factor 1 (HIF-1) signaling, autophagy, retinoic acid-inducible gene I (RIG-I) signaling, Toll-like receptor signaling, fatty acid oxidation/degradation, and IL-17 signaling. All these pathways can be either hijacked or suppressed by the viral proteins, leading to improved viral survival and life cycle. Aberration in pathways such as HIF-1 signaling and relaxin signaling in the lungs suggests the pathogenic lung pathophysiology in COVID-19. From enrichment analysis, it was evident that the deregulated genes in SARS-CoV-2 infection might also be involved in heart development, kidney development, and AGE-RAGE signaling pathway in diabetic complications. Anomalies in these pathways might suggest the increased vulnerability of COVID-19 patients with comorbidities. Moreover, we noticed several presumed infection-induced differentially expressed transcription factors and epigenetic factors, such as miRNAs and several histone modifiers, which can modulate different immune signaling pathways, helping both host and virus. Our modeling suggests that SARS-CoV-2 integrates its proteins in different immune signaling pathways and other cellular signaling pathways for developing efficient immune evasion mechanisms while leading the host to a more complicated disease condition. Our findings would help in designing more targeted therapeutic interventions against SARS-CoV-2.

Abstract 442: Generation of an Abcg1 Knock Out Mouse on the Reversa Background

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Gillian Douglas ◽  
Lucy Trelfa ◽  
Keith Channon ◽  
Ben Davies ◽  
Shoumo Bhattacharya
Keyword(s):  
Amino Acid ◽  
Intima Media Thickness ◽  
Null Alleles ◽  
Cholesterol Transport ◽  
Loss Of Function ◽  
Phosphate Binding ◽  
Knock Out ◽  
Plaque Regression ◽  
Genomic Editing

Background: Clinically HDL mediated reverse cholesterol transport (RCT) from macrophages has been shown to be inversely associated with carotid intima media thickness. Cholesterol efflux to mature HDL is mediated by ATP binding cassette transporter G1 (Abcg1). Abcg1 pays a key role in cholesterol transport with loss of function in macrophages and endothelial cells associated with significant cholesterol accumulation. However, mechanistic studies into the role of Abgc1 in plaque regression have been restricted due to the limitations of current regression models. Aims: To use TALENS mediated genomic editing to generate an Abcg1 knockout mouse on the REVERSA background to enable the investigation of its role in plaque regression. Methods and results: TALENs constructs were targeted to exon 3 upstream of the phosphate binding Walker A domain. TALEN mRNA was injected into REVERSA oocytes which were then implanted into foster mice. Founders were screened by Cel1 nuclease assay and sequencing. Three independent alleles were identified two of which create frameshift mutations (predicted to be null alleles) and one which resulted in a 3 amino acid deletion and a one amino acid substitution near the Walker A domain (potential hypomorphic allele). The two founder lines with frame shift mutations (KO 145 and 171) were taken forward for additional analysis. RNA extracted from primary macrophages from WT (REVERSA) and homozygous Abcg1 knockout mice was used to confirm the mutation was transcribed to RNA. Intron-spanning primers were designed and a product of the expected size was obtained and sequence analysis confirmed the insertion (KO-145) and deletion (KO-171) within the WALKER A domain of Abcg1. To ensure the mutations resulted in loss of function, a radioactive RCT assay was carried out in bone marrow derived primary macrophages. A significant decrease in RCT to HDL was observed in macrophages from both the KO-145 and KO-171 lines as expected there was no difference in RCT to ApoA1-I. Conclusions: Using genomic editing we have generated a gene specific knockout on the REVERSA background which will enable, for the first time, the investigation of the role of Abcg1 in plaque regression.

scholarly journals Rare mutations of ADAM17 from TOFs induce hypertrophy in human embryonic stem cell-derived cardiomyocytes via HB-EGF signaling

2019 ◽  
Vol 133 (2) ◽  
pp. 225-238 ◽  
Author(s):  
Yifang Xie ◽  
Anyun Ma ◽  
Boshi Wang ◽  
Rui Peng ◽  
Yingchun Jing ◽  
...  
Keyword(s):  
Growth Factor ◽  
Heart Development ◽  
Heart Defects ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Heparin Binding ◽  
Loss Of Function ◽  
Knock Out ◽  
Critical Pathways

Abstract Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.

scholarly journals Lysine Bioavailability in School-Age Children Consuming Rice Is Reduced by Starch Retrogradation

2020 ◽  
Vol 150 (12) ◽  
pp. 3208-3215
Author(s):  
Katia Caballero ◽  
Ronit Mandal ◽  
Anubhav Pratap-Singh ◽  
David D Kitts ◽  
Ronald O Ball ◽  
...  
Keyword(s):  
Amino Acid ◽  
Repeated Measures ◽  
Energy Requirement ◽  
School Age Children ◽  
Rice Starch ◽  
Acid Oxidation ◽  
School Age ◽  
Repeated Measures Design ◽  
Starch Retrogradation ◽  
The Impact

ABSTRACT Background Rice is one of the most commonly consumed cereal grains and is part of staple diets in the majority of the world. However, it is regarded as an incomplete protein, with lysine being a limiting amino acid. Objectives Our objectives were to determine the bioavailability of lysine in school-age children consuming cooked white rice and to assess the effect of rice starch retrogradation. Methods Bioavailability or metabolic availability (MA) of lysine was determined using the indicator amino acid oxidation (IAAO) method in a repeated-measures design. Six healthy school-age children (3 boys, 3 girls) with a mean ± SD age of 6.8 ± 0.98 y randomly received 4 crystalline l-lysine intakes (2, 6, 10, 14 mg · kg−1 · d−1), and 5 rice intakes to provide lysine at 8, 11, or 14 mg · kg−1 · d−1. The 14 mg · kg−1 · d−1 intakes were measured twice as warm rice and once as cold rice (to assess the impact of starch retrogradation on MA). Diets provided protein at 1.5 g · kg−1 · d−1 and calories at 1.7 times the participant's measured resting energy requirement, and were isonitrogenous. Breath samples were collected at baseline and during an isotopic steady state for 13C enrichment measurement. The MA of lysine from rice was determined by comparing the IAAO response of rice with l-lysine using the slope-ratio and single intake methods. Starch retrogradation was characterized using differential scanning calorimetry. Results MA of lysine in warm rice measured in school-age children was 97.5% and was similar to a repeated rice study (97.1%) within the same study population. MA of lysine was reduced significantly (P < 0.05) to 86.1% when the cooked rice was consumed cold, which corresponded to detectable starch retrogradation. Conclusions To our knowledge, this is the first study to measure the MA of lysine from rice in school-age children. Although the bioavailability of lysine from rice is high, it can be reduced by retrogradation of its starch component. This trial was registered at clinicaltrials.gov as NCT04135040.

scholarly journals Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs

2020 ◽  
Vol 10 (3) ◽  
pp. 1029-1037 ◽  
Author(s):  
Brian H. Kim ◽  
GuangJun Zhang
Keyword(s):  
Protein Translation ◽  
Loss Of Function ◽  
Mrna Transcription ◽  
Cell Stage ◽  
Knock Out ◽  
Dna Mutation ◽  
Guide Rna ◽  
Genetic Tool ◽  
Dna Mutations ◽  
The Impact

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes (rnf185 and rnf215) were successfully mutated in F1 fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear.

Abstract P331: Upregulated TBX1 by genetic variants are associated with human congenital heart disease

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Liwei Yu ◽  
Binbin Li ◽  
Hongyan Wang
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Genetic Variants ◽  
Heart Development ◽  
Congenital Heart ◽  
Minor Allele ◽  
Expression Level ◽  
Cardiac Progenitors ◽  
Rare Mutation ◽  
The Impact

Congenital heart disease (CHD) is the most common human birth defect worldwide. The cause of CHD is so far not well understood. Uncovering genetic factors leading to CHD is still a pressing task to be solved. TBX1 is one of the transcription factors early expressed in embryonic cardiac progenitors. In animal models, imbalanced TBX1 activity leads to cardiac defects. Given the dosage effect of TBX1, it is possible that genetic variant altering TBX1 function or expression level would affect heart development and contribute to CHD. In order to study the association of genetic variants of TBX1 and CHD susceptibility, we performed genetic screening in 409 CHD patients and 213 healthy controls. Bioinformatic and in vitro functional studies were performed to evaluate the impact of genetic variants. One single nucleotide polymorphism (SNP), rs41260844, in TBX1 promoter region was identified to be associated with CHD. The minor allele of rs41260844 is associated with higher CHD risk and shows increased TBX1 promoter activity (Fig A). Further study showed the minor allele attenuates TBX1 promoter binding affinity with nuclear protein(s) (Fig B). In addition, a novel case-specific missense rare mutation, p.P164L, in T-box domain was identified and predicted as a deleterious mutation. Functional analysis showed a trend of increased TBX1 function with the rare mutation. In summary, we concluded that a higher TBX1 expression level or activity is associated with CHD susceptibility, which could affect TBX1 downstream targets and thus disrupt the balance of the complex regulation network during cardiogenesis.

Sign in / Sign up

Export Citation Format

Share Document