title: NMR spectroscopic characterization of sarcolemmal permeability during myocardial ischemia and reperfusion.
abstract: This study aims to characterize the pattern of membrane disintegration during myocardial ischemia and reperfusion. Intracellular volumes were measured by 1H and 59Co NMR in isolated rat hearts during 10, 30 and 60 min of total ischemia and 30 min of reperfusion at normothermia. Perfusion with hypo-osmotic medium (210 mosm/l) increased intracellular water from 2.50+/-0.06 to 3.07+/-0.07 ml/g dry weight (P<0.001) during pre-ischemia. Hypo-osmotic swelling decreased by 16+/-3, 32+/-6 and 44+/-11% of the pre-ischemic value after 10, 30 and 60 min of ischemia (n.s., P<0.005, P<0.001) respectively, indicating that membrane permeabilization facilitated efflux of osmolytes and counterbalanced the osmotic driving force for water influx. Hypo-osmotic swelling decreased during 30 min of reperfusion by 18+/-5% in all groups (P<0.0.005 v post-ischemia). The volume of distribution of the extracellular marker cobalticyanide increased by more than 3.2+/-0.4 and 5.8+/-0.5% of the intracellular space after 30 and 60 min of ischemia respectively (P<0.001), and by an additional 2% after reperfusion. During 30 min of reperfusion, hearts released 1.6+/-0.2 and 3.2+/-0.4% of the intracellular creatine kinase contents after 30 and 60 min of ischemia, respectively (P<0.001). In addition to the correlation between ischemia duration and membrane permeability, evident from the analysis of each probe, the data showed a progressive increase in severity of membrane injury over time and permeabilization to larger molecules. 23Na NMR spectroscopy in conjunction with an extracellular shift reagent (SR) showed formation of a resonance at an intermediate chemical shift in between the intra and extracellular Na+ peaks, suggesting penetration of SR into cells with disrupted membranes. The constant chemical shift and narrow line shape of this resonance, characteristic of a homogeneous chemical environment, suggested that the distribution of SR was contained within the cytosol of cardiomyocytes. We propose that sarcolemmal membranes are gradually permeabilized to larger molecules by ischemia, and the evolving chemical instability is spatially contained within the myocyte.
authors.affiliation: Center for Light Microscope, Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, PA 15213, USA. askenasy+@andrew.cmu.edu
authors.forename: N
authors.initials: N
authors.lastname: Askenasy
authors.forename: A
authors.initials: A
authors.lastname: Vivi
authors.forename: M
authors.initials: M
authors.lastname: Tassini
authors.forename: G
authors.initials: G
authors.lastname: Navon
authors.forename: D L
authors.initials: DL
authors.lastname: Farkas
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002463:Cell Membrane Permeability; D048429:Cell Size / Q000502:physiology; D003402:Creatine Kinase / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism / Q000502:physiology; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D017202:Myocardial Ischemia / Q000378:metabolism*; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D012508:Sarcolemma / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013997:Time Factors
chemical_list: D012964:Sodium; D003402:Creatine Kinase