title: Direct sampling from muscle cross sections for electrophoretic analysis of individual mitochondria.
abstract: Muscle is a highly heterogeneous tissue. Practical approaches to sample selectively small regions of muscle cross sections would help to effectively utilize analytical techniques on muscle studies while taking into account tissue heterogeneity. In this report, semimembranosus muscle tissue cross sections were directly sampled and analyzed by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Prior to CE-LIF analysis, a small region in the muscle cross section was stained with 10-nonyl acridine orange (NAO) which is a mitochondrion-selective fluorescent probe known to form a stable complex with cardiolipin, a phospholipid found only in mitochondria. By micromanipulation, the injection end of the capillary was brought into contact with the tissue exhibiting fluorescently labeled mitochondria. Sampling from a region similar in size to the cross section of a single fiber was carried out by applying 11 kPa of negative pressure for 3 s. When an electric field of -200V/cm was applied, fluorescently labeled mitochondria electromigrated and were individually detected by postcolumn LIF detection. For each sample, the electropherogram displays a migration time window with a collection of narrow peaks. The collection of individual peak measurements is represented as a distribution of individual intensities related to cardiolipin content of mitochondria and a distribution of individual electrophoretic mobilities. Positioning the capillary injection end was sufficiently spatially accurate to deplete mitochondria in the sampled region upon repetitive injections. Treatment of a muscle cross section with a protease (trypsin) prior to mitochondria sampling resulted in a higher number of detected mitochondria, suggesting that one of the effects of this enzyme is a partial digestion of the muscles myofibrils, which eases the release of interfibrillar mitochondria entangled within these fibers. The protease treatment also resulted in changes to the electrophoretic mobility distribution of individual mitochondria, which may imply that partial digestion of proteins bound to the mitochondria contributes to the alteration in the electrophoretic mobility of mitochondria. The ability to sample a region as small as a single muscle fiber cross section and its direct CE-LIF analysis opens exciting possibilities for the direct analysis of muscle biopsies and mapping the mitochondrial electrophoretic properties in highly heterogeneous tissues.
authors.affiliation: Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
authors.forename: Hossein
authors.initials: H
authors.lastname: Ahmadzadeh
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Johnson
authors.forename: LaDora
authors.initials: L
authors.lastname: Thompson
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Arriaga
mesh_terms: D000609:Aminoacridines / Q000737:chemistry; D000818:Animals; D002308:Cardiolipins / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods; D016616:Cryoultramicrotomy; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D007834:Lasers; D008297:Male; D008931:Mitochondria, Muscle / Q000737:chemistry*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D051381:Rats; D011914:Rats, Inbred BN; D011916:Rats, Inbred F344; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
chemical_list: D000609:Aminoacridines; D002308:Cardiolipins; C041691:10-N-nonylacridinium orange; D014357:Trypsin