Chemicals and reagents
Sixteen isotope-labelled lipid mediator (LM) internal standards (IS) including arachidonic acid-d8 (AA-d8), 6-keto prostaglandin F1α-d4 (6-keto-PGF1α-d4), prostaglandin F2α-d4 (PGF2α-d4), prostaglandin E2-d4 (PGE2-d4), prostaglandin D2-d4 (PGD2-d4), thromboxane B2-d4 (TXB2-d4), leukotriene B4-d4 (LTB4-d4), leukotriene C4-d5 (LTC4-d5), 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid (5-HETE-d8), 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid (15-HETE-d8), and 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid (12-HETE-d8), platelet-activating factor C-16-d4 (PAF C-16-d4), tetranor-prostaglandin E metabolite-d6 (tetranor-PGEM-d6), oleoyl ethanolamide-d4 (OEA-d4), docosahexaenoic acid-d5 (DHA-d5), and eicosapentaenoic acid- d5 (EPA-d5), and eighteen LM standard compounds including 6-keto-PGF1α, PGF2α, PGE2, PGD2, PGA2, TXB2, LTB4, LTC4, 12(S)-hydroxyheptadecatrienoic acid (12-HHT), 9-hydroxy-10,12-octadecadienoic acid (9-HODE), 13(S)-hydroxyoctadecadienoic acid (13-HODE), 17,18-dihydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (17,18-DiHETE), 8-hydroxy-4Z,6E,10Z,13Z,16Z,19Z-docosahexaenoic acid (8-HDoHE), N-arachidonoylethanolamine (AEA), EPA, DHA, OEA and AA were purchased from Cayman Chemical Co. (Ann Arbor, MI). Formic acid (reagent grade, ≥ 95%) was obtained from Sigma–Aldrich (St. Louis, MO, USA). HPLC-MS grade acetonitrile, water, methanol, and ethanol were purchased from J.T. Baker (Phillipsburg, NJ, USA). Tri‐reagent from Molecular Research Center, Inc. (Cincinnati, OH, USA). High‐capacity cDNA reverse‐transcription kit from Applied Biosystems (Foster City, CA, USA). Mouse Signal Transduction Pathway Finder PCR Array. RT2 First Strand Kit. RT2 Real‐Time SYBR green/Rox PCR master mix from SABiosciences (Valencia, CA, USA). RNeasy Mini Kit from Qiagen (Valencia, CA, USA).
Animals and infection with malaria
Male mice from 4-5 months of age (Swiss Webster), weighing between 25-30g were infected with malaria parasites Plasmodium berghei (strain ANKA 2.34) and Plasmodium chabaudi (strain CR), uninfected mice used as controls of the experiments. Plasmodium berghei ANKA isolated from rats Grammomys surdates 2.34, has been maintained by Swiss Webster mice passage in the lab, and Plasmodium chabaudi in Balb/c mice, as described by Hoffmann et al. . For the infections, the mice were intravenously inoculated with identical rates (200 µl) frozen red cell with 25% parasitaemia of malaria parasites diluted in PBS. The parasitaemias were monitored daily by microscopic examination of blood smears stained with GIEMSA. Mice with parasitemia of approximately 35% were selected for the analyses, based on our previous findings , that these levels of parasitemia lead to muscle dysfunction, but with no signs of brain dysfunction (i.e. deviation of head, seizures and coma followed by death). Therefore, in this study, only the mice without symptoms of cerebral malaria were used. These levels of parasitaemias were achieved 3-6 days after injection of parasites. Mice were euthanized by cervical dislocation and then intact muscles (gastrocnemius) were isolated, kept stabilized in RNA later and shipped to University of Texas at Arlington.
RNA isolation and RT‐PCR gene arrays
The Mouse Signal Transduction Pathway-Finder PCR Array from SABiosciences was used to simultaneously detect gene expression changes of 10 signaling pathways (see Table 1, and details in Results). Total RNA was extracted from the cells using the Tri reagent according to manufacturer's protocol, quantified in a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) by determining absorbance at 260 nm in triplicate. RNA purity was indicated by the A260/280 nm absorbance ratio of 1.9 to 2.1 and A260/230 nm absorbance ratio ≥1.8. Using 0.5 μg of RNA, each sample was reverse transcribed in a 20‐µL reaction volume. cDNA was synthesized using the RT2 First Strand Kit and the PCR Array was run according to the manufacturer’s protocol, including a threshold of 0.25 and validation of each gene tested by the identification of single peaks in melting curves. As above, data were analyzed using RT2 Profiler PCR Array Data Analysis Software (SABiosciences); CT values were normalized to built‐in six reference housekeeping genes, genomic DNA control, reverse transcription control, and positive PCR control . We used this analytical software to set the statistical significance of upregulation or downregulation of all tested genes at fivefold difference. A major advantage of this technology is the pre-validation of these pathways at the protein level by the manufacturer and the robust utilization of internal and reference controls.
All components of LC-MS/MS system are from Shimadzu Scientific Instruments, Inc. (Columbia, MD, USA). LC system was equipped with four pumps (Pump A/B: LC-30AD, Pump C/D: LC-20AD XR), a SIL-30AC autosampler (AS), and a CTO-30A column oven containing a 2-channel six-port switching valve. The LC separation was conducted on a C8 column (Ultra C8, 150 × 2.1 mm, 3 µm, RESTEK, Manchaca, TX, USA) along with a Halo guard column (Optimize Technologies, Oregon City, OR, USA). The MS/MS analysis was performed on Shimadzu LCMS-8050 triple quadrupole mass spectrometer. The instrument was operated and optimized under both positive and negative electrospray and multiple reaction monitoring modes (+/− ESI MRM). The settings of flow rate and gradient program for the LC system as well as MS/MS conditions are recommended by a software method package for 158 lipid mediators (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA) and further optimized following our previously published quantification method . All analyses and data processing were completed on Shimadzu LabSolutions V5.91 software (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA).
Tissue preparation for lipidomic analyses
Striated muscles were isolated after mice sacrifice, then snap frozen in liquid nitrogen immediately and stored at -80°C. Before the experiment, the aliquoted frozen muscle tissue (50-100 mg) were defrosted on ice and in the dark, weighed carefully, and minced into small pieces on ice. The minced muscle was placed into a 2.0 mL round-bottom low retention microcentrifuge tube (Fisher Scientific, Waltham, MA) and 1.0 mL of ice-cold 80% methanol in water (v/v) will be added. The mixture was homogenized using a Tissue Lyser II homogenizer (Qiagen, Germantown, MD) at the frequency of 30 s-1, in 8×30-s bursts, waiting 20 s in between to avoid high temperature. The obtained homogenate was mixed with 5 µL of isotope-labelled LM internal standards (IS) mixture stock solution (5 µg/mL for AA-d8, 2 µg/mL for DHA-d5 and EPA-d5, and 0.5 µg/mL for the rest IS), and then agitated on ice and in the dark for 1-2 h, followed by centrifugation at 6000×g at 4°C for 10 min to remove any tissue residue and precipitated proteins.
All muscle samples need to be cleaned and concentrated by Solid Phase Extraction (SPE) before being injected into LCMS. Ice-cold 0.1% formic acid (4 mL) was added in the obtained supernatant to fully protonate the LM species before sample was loaded to the preconditioned SPE cartridges (Strata-X 33 µm polymeric reversed phase, Phenomenex, Torrance, CA, USA). Once the sample had been totally loaded, cartridges were washed with 0.1% formic acid followed by 15% (v/v) ethanol in water to remove excess salts. Then the LMs from the SPE sorbent bed were eluted by methanol. Solvents were removed using an Eppendorf® 5301 concentrator centrifugal evaporator (Eppendorf, Hauppauge, NY, USA). The dried extracts were stored at -80°C immediately for future LC-MS/MS analysis.
For lipidomic analyses data is presented as mean ± SD of all samples in multiple experiments. One-way ANOVA with post hoc Tukey's test (α = 0.05) was performed for data analysis. Differences were considered statistically significant at p < 0.05.