Oxygen consumption of mammalian myocardial cells in culture: measurements in beating cells attached to the substrate of the culture dish.

A Lucite attachment which permitted the measurement of oxygen consumption in cells in culture without manipulating the cells was constructed. The attachment fit over commercially available dishes for cell culture and had an oxygen electrode built into it. Oxygen uptake of cells in culture was thus measured. Cells were attached to the substrate of the culture dish during the measurements and could be observed in an inverted phase microscope. Cells did not show any morphological changes, e.g., cell shapes or beating rate in case of myocardial cells, before and after the measurements of oxygen consumption. Using this method the rate of oxygen consumption was determined in rat myocardial and heart non-muscle cells in culture and also in HeLa and L6 cell lines. Myocardial cells in culture had an approximately four times higher rate of oxygen uptake compared with heart non-muscle, HeLa, and L6 cells. The oxygen uptake of beating myocardial cells was higher by about 50% compared with quiescent myocardial cells.

On-line calorimetry in the arterially perfused rabbit interventricular septum.

A method has been developed to measure myocardial heat production simultaneously with mechanical (developed tension, rate of contraction and relaxation) and metabolic parameters in the arterially perfused interventricular septum of the rabbit. The system allows control of rest tension, frequency of contraction, temperature, and composition of the perfusate. The technique is based on the differential measurement of the heat flux from the muscle to the calorimetric bath. This technique is able to resolve changes in heat production as small as 0.06 mW. The resting heat production measured with the present calorimeter (1.62 +/- 0.1 mW/g wet tissue) agrees with that obtained with thermopiles and with that calculated from measurements of oxygen consumption. The heat per contraction (6.5 +/- 1.4 mJ/g wet tissue) also agreed with that measured with thermopiles in rabbit papillary muscles. The heat production measured at 22 degrees C under severe hypoxia can be fully explained by the addition of the expected change of enthalpy due to the reaction 0.5 glucose-lactate, calculated on the basis of the lactate measured in the perfusate, and the expected change of enthalpy of oxygen consumption [assuming that all remaining O2 in perfusate (0.09 vol%) is used for combustion of glucose]. These results clearly demonstrated the feasibility of this method for the correlation of changes in energy turnover, through the measurement of the heat production, with mechanical and metabolic processes on-line in arterially perfused septum.

Perturbation measurements of papillary muscle elasticity.

Small step-length perturbations (less than 0.5% Lmax) have been applied to rabbit papillary muscle for the purpose of measuring muscle stiffness at rest and during isometric twitch-force development. These stiffness-force measurements were fitted to four mechanical analogs to evaluate whether any of the models adequately predict the mechanical properties of papillary muscle. Stiffness-force relations have been measured at varied initial muscle lengths so that a broad range of applicability of an analog could be evaluated. This study shows that none of the four experimentally definable analogs predicts the stiffness-force responses of the muscle over a physiological range of initial muscle lengths. A two-segment nonhomogeneous analog has the same stiffness-force characteristics as a six-parameter lumped model but cannot be differentiated from the lumped model without a measurement of segmental variations. It is concluded that the passive elastic elements of papillary muscle cannot be controlled only by the manipulation of total muscle length so as to deduce cross-bridge mechanical properties from whole-muscle measurements.

Contractile force measured in unskinned isolated adult rat heart fibres.

A number of investigators have succeeded in preparing isolated cardiac cells by enzymatic digestion which tolerate external [Ca2+] in the millimolar range. However, a persistent problem with these preparations is that, unlike in situ adult ventricular fibres, the isolated fibres usually beat spontaneously. This spontaneity suggests persistent ionic leakage not present in situ. A preferable preparation for mechanical and electrical studies would be one which is quiescent but excitable in response to electrical stimulation and which does not undergo contracture with repeated stimulation. We report here a modified method of cardiac fibre isolation and perfusion which leaves the fibre membrane electrically excitable and moderately resistant to mechanical stress so that the attachment of suction micropipettes to the fibre is possible for force measurement and length control. Force generation in single isolated adult rat heart fibres is consistent with in situ contractile force. The negative staircase effect (treppe) characteristic of adult not heart tissue is present with increased frequency of stimulation. Isometric developed tension increases with fibre length as in in situ ventricular tissue.

42K exchange during myocardial ischemia.

42K exchange was studied before and after total ischemia in isolated, blood-perfused rabbit interventricular septa at 37 degrees C and 72 beats/min. Of 11 septa made ischemic for up to 45 min after 42K labeling to asymptotic values, 10 showed no decline in 42K as measured by tissue probe counts 5 min after reperfusion. Of these 10 septa, 7 showed a 1.4-14.2% increase in 42K counts on reperfusion. Three of four muscles reperfused after 60 min of ischemia showed progressive 42K losses. Of 14 septa previously labeled with 42K, 9 showed a parallel decrease in 42K-exchange rate constants as measured by tissue probe and effluent during washing out after 2-45 min of ischemia. In five other muscles a nonparallel decrease in rate constants as measured by tissue probe and effluent during washout indicated inhomogeneity of 45K exchange. These results indicated no persistent impairment in Na pump activity for 30-45 min of total ischemia. After ischemia for 45 min or less there was no consistent relationship between recovery of mechanical function and preservation of 42K content. After 60 min of ischemia, irreversible mechanical injury was associated with loss of tissue potassium.

Ischemia in isolated interventricular septa: mechanical events.

Isolated blood-perfused rabbit interventricular septa were adapted for studies of global ischemia by enclosure in a constant-humidity nitrogen atmosphere. During ischemia, developed tension (DT) and maximal rate of relaxation (-dP/dt) declined monoexponentially, lambda = 0.39 min-1 at 37 degrees C and 72 beats/min with a Q10 of 1.4 for DT and a Q10 of 1.9 for -dP/dt. After a 60- to 90-s delay the maximal rate of tension development (+dP/dt) declined at the same rate as DT. Time-to-peak tension (TPT) shortened immediately with ischemia but action potential duration shortened after 60-90 s. Calcium at a concentration of 5 mM slowed the rate of decline of +dP/dt to lambda = 0.26 min-1. Upon reperfusion after 10 min of ischemia the rates of recovery of DT, +dP/dt, and -dP/dt were similar, lambda = 0.21-0.23 min-1, and were not temperature dependent. The magnitude of recovery was 10-17% less at 37 degrees C than 28 degrees C. Potassium at a concentration of 10 mM did not alter the rate of decline of mechanical function, but significantly (P less than 0.01) increased the magnitude of mechanical recovery. The results suggest depletion and/or repletion of single compartments as the rate-limiting steps in ischemia and reperfusion.

Energy liberation and chemical change in frog skeletal muscle during single isometric tetanic contractions.

Recent data obtained from Rana temporaria sartorius muscles during an isometric tetanus indicate that the time-course of phosphocreatine (PC) splitting cannot account for the total energy (heat + work) liberation (Gilbert et al. 1971. J. Physiol. (Lond.) 218:)63). As this conclusion is important to an understanding of the chemical energetics of contraction, similar experments were performed on unpoisoned, oxygenated Rana pipiens sartorius muscles. The muscles were tetanized (isometrically) at 0 degrees C for 0.6, 1, or 5 s; metabolism was rapidly arrested by freezing the muscles with a specially designed hammer apparatus, and the frozen muscles were chemically analyzed. Comparable myothermal measurments were made on frogs from the same batch. Results of these experiments indicate: (a) The energy liberation parallels the PC and ATP breakdown with a proportionality constant of 10.7 kcal/mol; (b) comparably designed experiments with sartorius muscles of R. temporaria revealed that the ratio of energy liberation to PC splitting was significantly greater than that observed in R. pipiens sartorius muscles; (c) there is no systematic difference between experiments in which metabolism was arrested by the hammer apparatus and others using a conventional immersion technique.

Energetics of shortening muscles in twitches and tetanic contractions. II. Force-determined shortening heat.

The extra heat liberation accompanying muscular shortening, the force-determined shortening heat, is defined as the difference between the heat produced when shortening occurs and that produced in an isometric contraction developing the same amount of force and performing the same amount of internal work. Based on this definition, the initial energy production in twitches and tetanic contractions (E) is given by E = A + f (P, t) + alpha(F)x + W, where A is the activation heat, f(P, t), the tension-related heat (a heat production associated with the development and maintenance of tension), alpha(F)x, the force-determined shortening heat, and W, the external work. It is demonstrated that this equation accurately accounts for the time-course of heat evolution and the total initial energy production in both twitches and tetani at 0 degrees C. The force-determined shortening heat is liberated, during shortening, in direct proportion to (a) the distance shortened, and (b) the force against which shortening occurs. The normalized value of the force-determined shortening heat coefficient, alpha(F)/P(o), is the same in both the twitch and the tetanus. Finally, this formulation of the muscle's energy production also accounts for the total energy production in afterload isotonic twitches at 20 degrees C, where a Fenn effect is not demonstrable.

Activation heat, activation metabolism and tension-related heat in frog semitendinosus muscles.

1. Frog semitendinosus muscles were stretched to various lengths beyond the rest length (l(0)) and their initial heat and isometric tension production were measured.2. As the overlap between the thick and thin filaments is reduced, the initial twitch heat and tension decline in a linear manner. At a point at which the twitch tension approaches zero, the initial heat is 30% of that seen at l(0). It is concluded that this heat is the activation heat and reflects the energetics of calcium release and reaccumulation. The initial heat at shorter sarcomere lengths appears to be the sum of the activation heat plus a heat production associated with the interaction of the thick and thin filaments.3. A similar relationship between heat and tension production is seen in tetanic contractions.4. The time course of activation heat production in a twitch can be resolved into two phases: a temperature insensitive (Q(10) < 1.3) ;fast' phase (with a time constant of 45 msec) and a temperature sensitive (Q(10) = 2.8) ;slow' phase (with a time constant of 330 msec at 0 degrees C).5. Measurements of the creatine phosphate (PC) hydrolysis by muscles contracting isometrically at various muscle lengths at and beyond l(0), indicate an enthalpy change of -11.2 kcal/mole PC hydrolysed. The enthalpy change for the ATP hydrolysis by muscles stretched so that little or no tension was produced with stimulation was -9.9 kcal/mole ATP hydrolysed. It is concluded that the net activation heat is produced by the hydrolysis of PC or ATP.

Energetics of cardiac contractions.

1. The average resting heat production of a muscle under zero tension is 24.8 mcal/g muscle .min at 20 degrees C. In the majority of muscles examined the resting heat production increases when the resting tension and muscle length are increased.2. The relation between actively developed tension and heat produced is similar to that existing in skeletal muscle. The plot of heat against developed tension can be obtained either by altering muscle length or by varying the stimulus frequency.3. The mean maximum total efficiency work/(work + heat) in the work experiments was 11.6%. The total energy produced (work + heat) correlates with the load rather than with the work done.4. In isotonic contractions more heat is liberated than the heat versus tension plot predicts. This extra heat is load-dependent.

Activation heat in frog sartorius muscle.

Upon excitation of a muscle with two stimuli and variation of the interval between them up to the end of the period of full mechanical fusion, an increment of the isometric heat over that found in a single twitch is obtained. This is a good approximation to the activation heat, directly at 0 degrees C, or after certain corrections which become important at higher temperature. The activation heat so found is independent of the muscle length and nearly independent of temperature. It is increased by nitrate and caffeine.