Summary of Study ST002056

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001301. The data can be accessed directly via it's Project DOI: 10.21228/M8JH7P This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST002056
Study Title	Integrated Multilayer Omics Reveals the Genomic, Proteomic and Metabolic Influences of the Histidyl Dipeptides on Heart
Study Type	Triomics
Study Summary	Histidyl dipeptides, such as carnosine, present in a micro-millimolar ranges in the heart, are synthesized via the enzyme carnosine synthase (Carns). These dipeptides facilitate glycolysis by proton buffering, form conjugates with reactive aldehydes, such as acrolein, and attenuate ischemia and reperfusion injury. While these dipeptides exhibit multifunctional properties, a composite understanding of their roles in myocardium is lacking. To identify the landscape of histidyl dipeptide mediated responses in the heart, we used a triomics approach of genome wide RNA sequencing, global proteomics and unbiased metabolomics in the cardio specific Carns transgenic (Tg) mice and integrated the three data sets. Our result show higher myocardial levels of histidyl dipeptides lead to extensive changes in several microRNAs, which could target the expression of contractile proteins, beta-fatty acid oxidation and citric acid cycle (TCA) enzymes. Global proteomics shows, expression of contractile proteins, enzymes of beta-fatty acid oxidation and TCA cycle, were enriched in the CarnsTg heart. Under aerobic conditions, the CarnsTg hearts had lower levels of short and long-chain fatty acids and TCA cycle intermediate-succinic acid, whereas, under ischemic conditions the accumulation of fatty acids and TCA cycle intermediates were significantly attenuated in the CarnsTg heart. Integration of multiple data sets suggests that beta-fatty acid oxidation and TCA cycle pathways exhibited correlative changes in the CarnsTg hearts at all three levels. Our triomics approach shows histidyl dipeptides are critical regulators of myocardial structure, function and energetics.
Institute	University of Louisville
Last Name	Baba
First Name	Shahid
Address	580 S. Preston St
Email	spbaba01@louisville.edu
Phone	5028524274
Submit Date	2021-12-15
Num Groups	4
Analysis Type Detail	GC-MS
Release Date	2022-01-31
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002144
Sampleprep Summary:	To extract polar metabolites, 800 µL of methanol was added to 200 µL of homogenized heart sample. Mixture was vortexed for 2 min and then placed on ice for 10 min, followed by another 2 min of vortex mixing, and centrifuged at 15,000 rpm for 20 min at 4 °C. Supernatant was transferred into a glass vial and dried in a SpeedVac evaporator to remove methanol, followed by lyophilization to remove water. The dried metabolite extract was dissolved with 30 µL of 20 mg/mL methoxyamine hydrochloride pyridine solution followed by vigorous vortex mixing for 1 min. Methoxylation was performed by sonicating the sample for 20 min and incubation at 60 °C for 1 h. Derivatization was performed by adding 20 µL of N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) or N-Methyl-N-tert-butyldimethysilyltrifuoroacetamide (MTBSTFA) with 1% trimethylchlorosilane to the glass vial. Samples were incubated for 1 h at 60 °C and the mixture was transferred to a GC vial for analysis. Pooled samples were prepared by mixing 30 µL of derivatized metabolite extract from each sample to monitor the instrumental variations during the course of GC×GC-MS analysis.

Sampleprep ID:

SP002144

Sampleprep Summary:

To extract polar metabolites, 800 µL of methanol was added to 200 µL of homogenized heart sample. Mixture was vortexed for 2 min and then placed on ice for 10 min, followed by another 2 min of vortex mixing, and centrifuged at 15,000 rpm for 20 min at 4 °C. Supernatant was transferred into a glass vial and dried in a SpeedVac evaporator to remove methanol, followed by lyophilization to remove water. The dried metabolite extract was dissolved with 30 µL of 20 mg/mL methoxyamine hydrochloride pyridine solution followed by vigorous vortex mixing for 1 min. Methoxylation was performed by sonicating the sample for 20 min and incubation at 60 °C for 1 h. Derivatization was performed by adding 20 µL of N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) or N-Methyl-N-tert-butyldimethysilyltrifuoroacetamide (MTBSTFA) with 1% trimethylchlorosilane to the glass vial. Samples were incubated for 1 h at 60 °C and the mixture was transferred to a GC vial for analysis. Pooled samples were prepared by mixing 30 µL of derivatized metabolite extract from each sample to monitor the instrumental variations during the course of GC×GC-MS analysis.