Abstract
Previous studies have shown that glycolysis can oscillate periodically, driven by feedback loops in regulation of key glycolytic enzymes by free ADP and other metabolites. Here we show both theoretically and experimentally in cardiac myocytes that when the capacity of oxidative phosphorylation and the creatine kinase system to buffer the cellular ATP/ADP ratio is suppressed, glycolysis can cause large scale periodic oscillations in cellular ATP levels (0.02-0.067 Hz), monitored from glibenclamide-sensitive changes in action potential duration or intracellular free Mg2+. Action potential duration oscillations originate primarily from glycolysis, since they 1) occur in the presence of cyanide or rotenone, 2) are suppressed by iodoacetate, 3) are accompanied by at most very small mitochondrial membrane potential oscillations, and 4) exhibit an anti-phase relationship to NADH fluorescence. By uncoupling energy supply-demand balance, glycolytic oscillations may promote injury and electrophysiological heterogeneity during acute metabolic stresses, such as acute myocardial ischemia in which both oxidative phosphorylation and creatine kinase activity are inhibited.
Highlights
Previous studies have shown that glycolysis can oscillate periodically, driven by feedback loops in regulation of key glycolytic enzymes, such as phosphofructokinase (PFK),3 by free ADP, ATP, and other metabolites [1, 2]
We hypothesized that glycolytic oscillations would be most likely to occur when the capacity of oxidative phosphorylation and the creatine kinase (CK) system to buffer the cellular free ATP/ADP ratio is limited, conditions that may occur during acute myocardial ischemia [14, 15,16,17]
We have presented theoretical and experimental evidence from patch clamped isolated rabbit ventricular myocytes demonstrating that glycolysis can oscillate when the capacity of oxidative phosphorylation to buffer the cellular free ATP/ADP ratio, assisted by the CK system, becomes compromised
Summary
Cell Isolation—Ventricular myocytes were enzymatically isolated from the hearts of 2–3-kg rabbits, as described previously [23]. The pipette solution contained 140 mM KCl, 1 mM MgCl2, 10 mM 1,2-bis(2-aminophenoxy)ethaneN,N,NЈ,NЈ-tetraacetic acid, and 10 mM HEPES (pH 7.2 with Tris), to which fluorescent dyes were added as described below. Fluorescence Imaging—To image mitochondrial membrane potential, cells were superfused with Tyrode’s solution containing 30 nM tetramethylrhodamine methyl ester (TMRM) for at least 30 min. To image intracellular free Mg2ϩ, myocytes were loaded with 100 M pentapotassium salt of Mg Green through the patch pipette, and imaging was performed using a 480/530-nm band pass filter for excitation/ emission, respectively. Mg Green is a low affinity Ca2ϩ indicator (Kd ϭ ϳ6 M), under our usual experimental conditions with 10 mM 1,2-bis(2-aminophenoxy)ethaneN,N,NЈ,NЈ-tetraacetic acid in the patch pipette solution, changes in fluorescence intensity mainly reflected change in free intracellular [Mg2ϩ] [24]. The differential equations were numerically solved by a fourth order RungeKutta method with a time step of 0.0005 s
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