IMPAIRED POSTHYPOXIC RELAXATION IN SINGLE CARDIAC MYOCYTES - ROLE OF INTRACELLULAR PH AND INORGANIC-PHOSPHATE

Objective: The aim was to examine the effects of alterations in intrac ellular pH and inorganic phosphate concentration (known to influence m yofilament kinetics and to change rapidly during hypoxia) on cell cont raction relaxation, and the Ca'' transient in normoxic and hypoxic myo cytes. Methods: Single adult rat ventricular myocytes were electricall y stimulated (0.2 Hz) and cell length (photodiode array), intracellula r Ca2+ (indo-I fluorescence), or intracellular pH (SNARF-I fluorescenc e) measured. Hypoxia was induced in a special open chamber in which a laminar layer of argon prevented the back diffusion of atmospheric oxy gen. Results: Electrically stimulated contraction was preserved during exposure to hypoxia. At reoxygenation 10 minutes later the time from the stimulus to the peak of contraction (TPK) increased by 30(SEM 9)% and the time from the peak of contraction to 50% recovery of cell leng th (RT50) increased by 59(13)% relative to prehypoxic values (n = 8). These changes were not accompanied by a change in the kinetics of the Ca2+ transient. pH(i) fell from a baseline of 7.33(0.04) to 7.25(0.03) during hypoxia and then overshot to 7.44(0.03) at reoxygenation (n = 5). Since an intracellular alkalosis can slow myofilament relaxation, proton extrusion routes were blocked to examine posthypoxic relaxation in the absence of an alkalosis. Despite inhibition of the pH(i) overs hoot, posthypoxic relaxation remained impaired. Intracellular inorgani c phosphate levels were manipulated in two protocols (2-deoxyglucose t o ''trap'' phosphate and Tris(hydroxymethyl)-aminomethane to buffer ph osphate) and both TPK and RT50 increased in normoxic cells. Having est ablished that these two interventions, which would be expected to decr ease intracellular inorganic phosphate, result in a slowing of relaxat ion, myocytes were first phosphate loaded (exposed to 5.0 mM phosphate ) and then made hypoxic and reoxygenated after 10 min to blunt the exp ected fall in phosphate accompanying reoxygenation. This led to a redu ction in the slowing of contraction and relaxation following reoxygena tion [TPK increased by 7(5)% and RT50 by 17(9)%, n = 8; p < 0.05 v cel ls studied in control buffer]. Conclusions: Impaired posthypoxic relax ation is not the result of changes in pH(i) but is attenuated by phosp hate loading of cells and may be due to a rapid decrease in intracellu lar phosphate accompanying the resynthesis of high energy phosphates a t reoxygenation.