Percent fractional shortening ( FS), which quantifies contraction of the ventricular wall and is definitely an indication of muscle function, was calculated as FS = ([LVDd – LVDs] / LVDd) ?one hundred. RNA isolation and gene expression evaluation Total RNA was extracted from the heart using the phenol guanidine isothiocyanate method (TRIZOL kit, Invitrogen, Carlsbad, CA) as per the manufacturer’s directions. Total RNA (0.7 ) was reverse transcribed for 1h at 50 using the Thermoscript RT-PCR technique (Invitrogen, Carlsbad, CA), which was then amplified applying an iCycler (Biorad) with Brilliant SYBR Green QPCR master mix (Stratagene, Palo Alto, CA, USA). Relative quantification of gene expression was normalized to 18S rRNA in MHC-ACS1 and MHCPPAR mice (primers are listed. See Table, Supplemental Digital Content material two). Plasma lipid measurements Blood from overnight fasted mice was collected in the retro-orbital plexus to measure plasma total cholesterol (TC), triglycerides (TG), FAs and glucose. For measurement of myocardial TG, hearts have been perfused with PBS and homogenized at four in 1 M NaCl buffer containing lipase inhibitors to stop TG hydrolysis. Lipids have been extracted from heart tissues (50 mg) based on solutions modified from Folch et al. (16). Dried lipids have been solubilized in PBS containing two Triton X-100. Heart TG, plasma TG, TC and absolutely free FAs have been measured enzymatically utilizing Infinity kits (Thermo Electron Corp, Middletown, VA) plus a NEFA C kit (Wako, Osaka, Japan). Plasma glucose was measured by Autokit Glucose (Wako, Osaka, Japan). Cardiac acyl CoA, ceramide and diacylglycerol (DAG)NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptExtraction of myocardial acyl CoAs, DAG and ceramides were performed as described previously (17, 18).1421473-07-5 web Also, for the extraction of ceramides, C12 and C25 ceramides have been spiked as internal standards.5-Chloro-3-methyl-1H-pyrazole web Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analyses Total cardiac acyl CoAs have been measured by LC/MS/MS as previously described (15, 17). Acyl CoAs and ceramides were measured on a Waters Xevo TQ MS ACQUITY UPLC technique (Waters, Milford, MA). Ceramide samples have been loaded onto a Waters ACQUITY UPLC BEH Phenyl column (three mm inner diameter ?one hundred mm with 1.PMID:24278086 7 particles), preceded by a guard column. The UPLC flow rate was 300 /min in a binary gradient mode with water and methanol each containing 0.two formic acid and 1mM ammonium formate. Constructive ESI-MS/MS mass spectrometry was performed as described previously (18). Every ceramide species (C14:0, C16:0, C18:1, C18:0, C20:four, C20:1, C20:0, C22:1, C22:0, C24:1 and C24:0) was measured by various reaction monitoring mode. Total ceramide wasJ Cardiovasc Pharmacol. Author manuscript; obtainable in PMC 2014 April 01.Khan et al.Pagecalculated as sum of individual species. LC/MS/MS for acyl CoA was performed as described previously (15).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptLC/MS/MS for DAG was performed working with a bench-top tandem mass spectrometer, API 3000 (PerkinElmer Life Sciences), interfaced having a TurboIonspray ionization source or atmospheric pressure chemical ionization supply. Peripherals integrated a PerkinElmer series 200 micro-pump and an autosampler. DAGs (derived from C16:1, C16:0, C18:0, C18:2, C18:1, C20:4, C22:five and C22:6) were ionized in good atmospheric pressure chemical ionization mode. [M+H-18]+/product ions from corresponding fatty acid moiety had been monitored for chosen reaction monitorin.