Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA063996

Local Sample ID:	#B10_0107 YKT_11/8/16_#94
Subject ID:	SU001058
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	FVB/N
Age Or Age Range:	16.5 to 18.5 weeks old
Weight Or Weight Range:	25 to 30 grams
Gender:	Male
Animal Animal Supplier:	in house breeding
Animal Feed:	Standard chow

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Subject:

Subject ID:	SU001058
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	FVB/N
Age Or Age Range:	16.5 to 18.5 weeks old
Weight Or Weight Range:	25 to 30 grams
Gender:	Male
Animal Animal Supplier:	in house breeding
Animal Feed:	Standard chow

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
#B10_0107 YKT_11/8/16_#94	SA063996	FL010931	Plasma	Tissue
#B10_0107 YKT_11/8/16_#94	SA063996	FL010931	TACSEV	Intervention

Collection:

Collection ID:	CO001052
Collection Summary:	At study endpoint, prior to tissue collection, mice were anesthetized with sodium pentobarbitone (Lethabarb, 80mg/kg). Once mice had reached an adequate level of anesthesia (assessed by testing the pedal reflex), blood was collected via cardiac puncture using a 25 G needle and 1 ml syringe. This procedure was followed by cervical dislocation. Collected blood was stored on ice in K2EDTA tubes (Beckton Dickinson), centrifuged at 4°C, 4000 g for 10 min, and plasma collected into fresh tubes and stored at -80°C.
Sample Type:	Blood (plasma)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001072
Treatment Summary:	Swim training - Physiological hypertrophy in mice was achieved via swim training twice daily for 4 weeks, as previously described (McMullen et al., 2003). Swim training commenced in adult mice at approximately 3 months of age with 10 min sessions on the first day, with 10 min increments each day until the maximum of 90 min per session was reached. Mice were rested for at least 4 hours between each session, and water temperature was maintained between 30 to 32°C to avoid thermal stress. At the end of every session, mice were individually towel dried to prevent hypothermia. Plasma and hearts were collected 3 h after the last swim session. Transverse aortic constriction - Male mice were subjected to either sham or TAC surgery at approximately 3-3.5 months of age, as previously described (Xu et al., 2008). Two variations of TAC were performed, designated TAC-moderate (TAC-MOD) and TAC-severe (TAC-SEV). Aortic banding performed on the TAC-MOD mice used a blunt 25 G needle (outside diameter 0.5 mm) placed next to the aorta during ligation to obtain a consistent degree of banding (constriction of blood vessel from ~1 mm to 0.5 mm), while a 27 G needle (outside diameter 0.4 mm) was used for the TAC-SEV group (constriction of blood vessel from ~1 mm to 0.4 mm). Sham operated mice were subjected to the same procedure but no ligation was made.

Treatment ID:

TR001072

Treatment Summary:

Swim training - Physiological hypertrophy in mice was achieved via swim training twice daily for 4 weeks, as previously described (McMullen et al., 2003). Swim training commenced in adult mice at approximately 3 months of age with 10 min sessions on the first day, with 10 min increments each day until the maximum of 90 min per session was reached. Mice were rested for at least 4 hours between each session, and water temperature was maintained between 30 to 32°C to avoid thermal stress. At the end of every session, mice were individually towel dried to prevent hypothermia. Plasma and hearts were collected 3 h after the last swim session. Transverse aortic constriction - Male mice were subjected to either sham or TAC surgery at approximately 3-3.5 months of age, as previously described (Xu et al., 2008). Two variations of TAC were performed, designated TAC-moderate (TAC-MOD) and TAC-severe (TAC-SEV). Aortic banding performed on the TAC-MOD mice used a blunt 25 G needle (outside diameter 0.5 mm) placed next to the aorta during ligation to obtain a consistent degree of banding (constriction of blood vessel from ~1 mm to 0.5 mm), while a 27 G needle (outside diameter 0.4 mm) was used for the TAC-SEV group (constriction of blood vessel from ~1 mm to 0.4 mm). Sham operated mice were subjected to the same procedure but no ligation was made.

Sample Preparation:

Sampleprep ID:	SP001065
Sampleprep Summary:	Cardiac tissue samples were homogenized and sonicated in phosphate buffered saline (PBS) (pH 7.4). The Bicinchoninic acid (BCA) assay (Thermo Scientific) was used to determine protein concentration. A mixture of internal standards (10µl) in CHCl3: methanol (1:1) and 200µl of CHCl3/methanol (2:1) were added to each sample (ventricles- 50µg protein in 10µl; plasma- 10µl) before being briefly vortexed. Samples were first mixed (rotary mixer, 10 min), sonicated (water bath, 30 min at room temperature [RT]) then rested at RT (20 min). Samples were centrifuged (16,000 g, 10 min) and the supernatant was dried under a constant stream of nitrogen gas at 40°C. Extracted lipids were resuspended in 50µl H2O saturated butanol then sonicated (10 min), before 50 µl of methanol with 10mM ammonium was added. Resuspended extracts were centrifuged (3,350 g, 5 min), and the supernatant transferred to glass vials with Teflon insert caps and stored at -80°C until analyzed. Lipid extracts were thawed at RT for an hour, then sonicated in a water bath for 15 min at RT before analysis.

Sampleprep ID:

SP001065

Sampleprep Summary:

Cardiac tissue samples were homogenized and sonicated in phosphate buffered saline (PBS) (pH 7.4). The Bicinchoninic acid (BCA) assay (Thermo Scientific) was used to determine protein concentration. A mixture of internal standards (10µl) in CHCl3: methanol (1:1) and 200µl of CHCl3/methanol (2:1) were added to each sample (ventricles- 50µg protein in 10µl; plasma- 10µl) before being briefly vortexed. Samples were first mixed (rotary mixer, 10 min), sonicated (water bath, 30 min at room temperature [RT]) then rested at RT (20 min). Samples were centrifuged (16,000 g, 10 min) and the supernatant was dried under a constant stream of nitrogen gas at 40°C. Extracted lipids were resuspended in 50µl H2O saturated butanol then sonicated (10 min), before 50 µl of methanol with 10mM ammonium was added. Resuspended extracts were centrifuged (3,350 g, 5 min), and the supernatant transferred to glass vials with Teflon insert caps and stored at -80°C until analyzed. Lipid extracts were thawed at RT for an hour, then sonicated in a water bath for 15 min at RT before analysis.

Combined analysis:

Analysis ID	AN001667
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 6490
Column	Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6490 QQQ
Ion Mode	POSITIVE
Units	pmol/umol PC

Chromatography:

Chromatography ID:	CH001174
Instrument Name:	Agilent 6490
Column Name:	Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001542
Analysis ID:	AN001667
Instrument Name:	Agilent 6490 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE