On the state of creatine in heart muscle.

It is well known'-4 that a considerable fraction of the total creatine in heart muscle is present in such a form that upon extraction by conventional procedures, such as trichloroacetic or perchloric acid, it will react directly with the diacetyl reagent. It is conventionally called the free creatine fraction and amounts to about 7 mM per kg of rabbit heart muscle.4 The concentration of free creatine in plasma is 0.03 mM per liter. There is therefore a steady-state difference in apparent concentration of 230 fold. Since creatine can be demonstrated to move across the muscle membrane, it would seem that there must either be a "pump" mechanism maintaining a steady-state concentration difference or the so-called free creatine must not all be actually in free solution in a compartment available for exchange with the extracellular fluids. The theoretical possibility that the membrane is virtually impermeable to creatine is discarded on the grounds that it is contrary to observation, and the possibility that there is a large source of creatine synthesis in the muscle cell is discarded also for lack of evidence for it. In order to investigate further the state of creatine in muscle, studies have been made using isotopically labeled creatine with isolated perfused rabbit hearts. Creatine-1-C'4 in tracer amounts has been incorporated in the perfusion fluid and the changes over time in the specific activity in the creatine phosphate and "free" creatine fractions in heart muscle measured. By such observations one can obtain information bearing upon the permeability of the cell membrane to creatine and upon the nature of the "free" creatine pool. Methods.-Male rabbits about 3 kg body weight were anesthetized with sodium pentobarbital (50 mgm/kg) and thoracotomized under endotracheal ventilation with oxygen. After heparin (300 U.S.P. units/kg), the aorta was cannulated and perfusion of the coronaries with oxygenated saline begun as quickly as possible to avoid myocardial hypoxia. The perfusate at this stage was discarded. The heart was transferred to a temperature-controlled chamber (370C) arranged for collection of the coronary venous outflow. After all blood was washed out, the venous outflow was returned to the reservoir system creating a closed circuit. The minimum volume used was 60 ml. The perfusion was at a pressure of 20 mm Hg maintained by finger-pump. The flow was about 25 ml per min. The perfusion fluid was a phosphate buffered solution, NaCl 145.5 KCl 2.7, CaCl2 1.8, KH2PO4 0.17, Na2HPO4 1.48, and glucose 5.56 mM per liter. It was aerated with 100% oxygen. The initial pH was about 7.6. After a period of closed circuit perfusion, CO2 and organic acids entered and the pH fell to 7.2-7.3. The exchanges of creatine were studied in hearts beating spontaneously, during prolonged arrest, and also after a previously arrested heart was returned to a rhythmic contractile state. Arrest was induced by shifting to a calcium-free perfusate and in addition, introducing rapidly into the flowing perfusion fluid entering the coronary vessels 3.5 ml of a 0.20% solution of sodium citrate. The citrate rich perfusate was discarded, as was the first 50 ml of calcium-free perfusion fluid. The heart was maintained in asystole so long as the perfusion fluid was calcium-free. The beat was restored by shifting to perfusion with normal calcium level. The details of the ion effects upon arrest and recovery are complex and will be presented in another report., Creatine-1-C'4 was added to the perfusion fluid at a concentration of 8.8 ,uM per 100 ml. The activity of the creatine was about 1.34 ,c/,4M. Since the entrance rate of creatine into the muscle cell is slow, it was essential to remove tracer-containing perfu~ate from the coronary