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We examined the effects of pharmacological alteration of Ca2+sources on mechanical and energetic properties of paired-pulse (“bigeminic”) contractions. The fraction of heat release that is related to pressure development and pressure-independent heat release were measured during isovolumic contractions in arterially perfused rat ventricles. The heat released by regular and bigeminic contractions showed two brief pressure-independent components (H1 and H2) and a pressure-dependent component (H3). We used the ratio of active heat (H′a) to pressure-time integral (PtI) and the ratio of H3 to PtI to estimate the energetic cost of muscle contraction (overall economy) and pressure maintenance (contractile economy), respectively. Neither of these ratios was affected by stimulation pattern. Caffeine (an inhibitor of sarcoplasmic reticulum function) significantly decreased mechanical responses and increased the energetic cost of contraction (Δ = 101 ± 12.6%). Verapamil (an L-type Ca2+channel blocker) decreased pressure maintenance of extrasystolic (Δ = 43.4 ± 3.7%) and postextrasystolic (Δ = 37.5 ± 3.5%) contractions without affecting postextrasystolic potentiation, suggesting that a verapamil-insensitive fraction is responsible for potentiation. The verapamil-insensitive fraction was further studied in the presence of lithium (45 mM) and KB-R7943 (5 μM), inhibitors of the Na+/Ca2+exchanger. Both agents decreased all mechanical responses, including postextrasystolic potentiation (Δ = 67.3 ± 3.3%), without altering overall or contractile economies, suggesting an association of the verapamil-insensitive Ca2+fraction to the sarcolemmal Na+/Ca2+exchanger. The effect of the inhibitors of the Na+/Ca2+exchanger on potentiation suggests an increased participation of extracellular Ca2+(and, thus, a redistribution of the relative participation of the Ca2+pools) during bigeminic contractions in rat myocardium.

Chagasic (Ch) and nonchagasic (NCh) IgG fraction (20 μg/ml) effects on cardiac performance of adult Wistar rat ventricles were studied with a novel approach applying a microcalorimetric technique. Resting heat ( Hr) was significantly decreased by Ch antibodies (Δ HrCh = 4.8 ± 0.9 mW/g). Although the Hr decrease can be associated with diminished activity of the Na+/K+ pump, the magnitude of the effect (25% of control Hr) indicates that additional processes may also be affected. Ch antibodies induced an initial increase in developed pressure (P), which was associated with a decreased contractile economy. However, after 30 min of Ch antibody perfusion, P reached a significantly lower level (ΔPCh = 3.8 ± 1.2 mN/mm2) without changes in active heat per beat ( Ha). Consequently, Ha/P ratio increased, indicating that the energetic cost per unit of P was higher. In contrast, P and Ha were both significantly and reversibly decreased by NCh antibodies (ΔPNCh = 4.4 ± 1.2 mN/mm2; Δ HaNCh = 9.7 ± 2.2 mJ/g), but Ha/P remained unaffected. According to these data, normal hearts exposed to Ch antibodies present a biphasic mechanical response: 1) an initial period of increased contractility (and decreased global muscle economy) consistent with antibodies with β1-adrenergic activity, such as those used in the present study, and 2) a decrease in P at 30 min of Ch antibody perfusion, which suggests that another Ca2+-related mechanism is compromised. These data contribute to redefine the role of antibody-mediated responses in the pathophysiology of chronic chagasic cardiomyopathy as agents of myocardial failure.

Fil: Bonazzola, Patricia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Cardiologicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiologicas; Argentina

Heat production under no-flow ischemia (ISCH) and under hypoperfusion (HYP) conditions was measured in single isovolumetric contractions of perfused rat ventricles at 25°C. Resting heat production (Hr) and resting pressure decreased when the perfusion rate was reduced from 6 to 1.5 mL·min-1 or lower flows (HYP) and by ISCH. Maximal developed pressure (P) decreased to 29% and 20% of control by HYP at 0.8 mL·min–1 and ISCH, respectively. The tension-independent heat (TIH) fraction attributed to Ca2+-binding, measured during single contractions, decreased under HYP with an increase in the ratio between the maximum relaxation rate and P (–P/P ratio). The TIH fractions (attributed to Ca2+ binding and Ca2+ removal processes) decreased under ISCH. The long duration TIH fraction associated with Ca2+-dependent mitochondrial activity disappeared at flow rates of 1.5 mL·min–1 or lower. The ratio between the tension-dependent energy release and P was decreased by ISCH but not by HYP, indicating that under ISCH there was an improvement in contractile economy, but this was not modified by HYP. Overall, the results indicate that no-flow and low-flow ischemias are energetically different models. While the contractile failure under HYP seems to be related to a decrease in myofilament Ca2+ sensitivity, under ISCH it appears to be related to decreased cytosolic Ca2+ availability combined with a more noticeable effect on a fraction of energy that has been attributed to mitochondrial activity. Furthermore, mechanical and energetic responses of both models (i.e., ISCH and HYP) found in the present work were not the same as those previously observed in severe hypoxia so that all these models should not be used indistinctly.Key words: calorimetry, hypoperfusion, ischemia, muscle economy, tension-dependent heat, tension-independent heat.

The consequences of an extrasystole (ES) on cardiac muscle’s energetics and Ca2+homeostasis were investigated in the beating heart. The fraction of heat release related to pressure development (pressure dependent) and pressure-independent heat release were measured during isovolumic contractions in arterially perfused rat ventricle. The heat release by a contraction showed two pressure-independent components (H1 and H2) of short evolution and a pressure-dependent component (H3). The additional heat released by ES was decomposed into one pressure-independent ([Formula: see text]) and one pressure-dependent ([Formula: see text]) component with time courses similar to those of control components H2 and H3. ES also induced the potentiation of pressure development (P) and heat release during the postextrasystolic (PES) beat. The slope of the linear relationship between pressure-dependent heat and pressure maintenance was similar in control, ES, and PES contractions (0.08 ± 0.01, 0.10 ± 0.02, and 0.08 ± 0.01 mJ ⋅ g−1⋅ mmHg−1⋅ s−1, respectively). The potentiation of H2 (heat component related with Ca2+removal processes) in PES was equal to [Formula: see text] at 0.3, 0.5, 1, and 2 mM Ca2+, suggesting that the extra amount of Ca2+mobilized during ES was recycled in PES. Pretreatment with 1 mM caffeine to deplete sarcoplasmic reticulum Ca2+content inhibited both the mechanical and energetic potentiation of PES. However, the heat released and the pressure developed during ES were not changed by sarcoplasmic reticulum depletion. The results suggest that 1) the source of Ca2+for ES would be entirely extracellular, 2) the Ca2+entered during ES is accumulated in the sarcoplasmic reticulum, and 3) the Ca2+stored by the sarcoplasmic reticulum during ES induces an increased contribution of this organelle during PES compared with the normal contraction.

Tension-dependent (TDH) and tension-independent heat (TIH) release were measured during single isovolumetric contractions in the arterially perfused rat ventricle. Under perfusion with 7 mM K-0.5 mM Ca, TDH showed only one component (H3), whereas TIH could be divided into two components (H1and H2) of short evolution (similar to the classically identified activation heat) and one component (H4) of long duration (dependent on mitochondrial respiration). Under 25 mM K, TIH components (i.e., H1, H2, and H4) increased with the increase in extracellular Ca concentration ([Ca]o) from 0.5 to 4 mM, and H3correlated with pressure at all [Ca]o, with regression parameters similar to those observed under 7 mM K. Under 25 mM K-2 mM Ca, peak pressure development (P), H1, H2, and H3, plotted against the number of beats under 0.4 μM verapamil, exponentially decreased, but H4decreased to 5.5 ± 2.9% in the first contraction and remained constant thereafter. Under hypoxia, P, H1, H2, and H3progressively decreased for about six contractions, but H4was not detectable from the second contraction. The results suggest that increasing extracellular K concentration decreases contractile economy mainly by increasing energy expenditure related to a Ca-dependent (verapamil-sensitive) mitochondrial activity that is not related to force generation.

Heat production and isovolumetric pressure development (P) were measured simultaneously in the arterially perfused rat ventricle. The time course of the calorimetric signal that follows a contraction could be decomposed into four components of energy released. Three of these components (H1, H2, and H4) were pressure independent, only H3 correlated with either P or the pressure-time integral (PtI) (r > 0.78, n = 36, P < 0.01). The dimensionless slope of the regression of H3 on P was 0.24 (an index of muscle economy) and the absence of O2 (N2 replacement) decreased it to 0.178 suggesting that 26% of H3 is related to oxidative metabolism. H4 was the most affected by the lack of O2 in the perfusate. It decreased to 16% in the first beat under N2 without change in P or in H1, H2 or H3, and disappeared (1.6 +/- 1.0 mJ.g-1) in the fourth contraction under N2 (while P, H1, H2 and H3 remained over 64% of their control values). H4 was activated during the first 1-3 beats after a quiescent period and remained active for several seconds (even in the absence of subsequent stimulation) as if the basal metabolism had been increased to a new steady level. H1 and H2 were dependent on the extracellular Ca. The magnitudes of both H1 (1.8 +/- 0.2 mJ.g-1) and H2 (2.7 +/- 0.2 mJ.g-1) were similar to those reported for the fast and slow components of activation heat in skeletal muscle. If twin stimuli are applied (200 ms apart), additional energy is released (+3.0 +/- 0.3 mJ.g-1) that can be decomposed in two components similar to those identified as H2 and H3. The magnitude of H1, its absence in the twin contraction and its Ca dependency suggest an association with Ca-binding processes (mainly Troponin C). The presence of an H2 component during the twin contraction, its magnitude and Ca dependence gives support to a relationship between H2 and Ca removal processes.

A new calorimetry method has been developed to measure heat production from heart cell suspensions under continuous perfusion. The method is technically independent of the temperature at which the measurements are made, allows full control of the perfusion media, and is suitable for various biological preparations such as cells from diverse tissues, membrane vesicles, or skinned cells. The resting heat rate (Hr) measured at 18.5 degrees C in three different species (19.2 +/- 0.43, 12.8 +/- 0.56, and 9.4 +/- 0.52 mW/g dry wt for rat, guinea pig, and rabbit ventricular myocytes, respectively) agrees with that obtained with other methodologies such as oxygen consumption, thermopiles, and whole heart calorimetry. The Hr measurements showed an excellent correlation with the percentage of rod-shaped cells, indicating that rounded cells are metabolically inactive. Although the time course of the effect of increasing extracellular [K] was dependent on the species, the new steady level of Hr observed under higher extracellular [K] was significantly higher in all three species (+8.3 +/- 1.2, +9.5 +/- 4.0, and +9.3 +/- 2.7 mW/g dry wt for rat, guinea pig, and rabbit ventricular cells, respectively). This indicates that the commonly used "arrested-heart" preparation (with high extracellular [K]) for evaluation of basal metabolism most probably overestimates the real resting values. The present results also show that the wide range of resting metabolism reported in whole tissue is not due to cellular heterogeneity nor to myocyte interaction and supports the idea of an inverse relationship between resting metabolism and body weight or animal size across species.

The mechanical and energetic effects of verapamil (VER) and reduction of extracellular Ca concentration ([Ca]o) were studied in the interventricular rabbit septa and the dog papillary muscle. Even though the negative inotropic effects of VER [i.e., decrease in developed tension (T), maximal rates of contraction (+T) and relaxation (-T), and tension time integral] qualitatively resemble [Ca]o reduction, VER also elicited an anti-relaxant effect (decrease in -T/T and prolongation of the last phase of relaxation) that was not found with [Ca]o reduction. Resting heat production was similar in both preparations and remained unaffected either by changes in [Ca]o or by the presence of VER. The ratio between T and active heat production per beat (H'a) under constant fiber length decreased with VER, and this decreased economy of contraction was more marked with the increase in contraction frequency. Conversely, the T/H'a remained unaltered with changes in [Ca]o. Tension-independent heat decreased in the presence of VER and, although muscle economy can be improved by increasing muscle length in a VER-treated muscle, it is not possible to achieve either the maximal T or the maximal contraction economy that can be obtained by stretching a nontreated muscle. It may be concluded that at constant fiber length and frequency of contraction VER decreases myocardial contractile force, impairs relaxation, and decreases contraction economy. Neither the mechanical nor the energetic effects of VER can be explained solely on the basis of a reduced extracellular Ca availability, so that either the density of the Ca that enters through the channel is different from that of other sources of Ca or VER has an effect on the cross-bridge cycling mechanism.

The role of calcium influx on energy expenditure during cardiac contraction was studied. For this purpose, the described ability of lithium and KB-R 7943 (KBR) to diminish Ca entry through Na-Ca exchanger (Ponce-HornosLanger, J Mol Cell Cardiol 1980, 12, 1367, Satoh et al., Circulation 2000, 101, 1441) were used. In isolated contractions (contractions elicited after at least 5 min of rest) LiCl 45 mmol L(-1) decreased pressure developed and pressure-time integral from 42.3 +/- 2.7 and 14.5 +/- 1.2 to 32.1 +/- 3.4 mN mm(-2) and 8.3 +/- 0.9 mN mm(-2) s, respectively. A similar effect was observed in regular contractions (at 0.16 Hz stimulation). The presence of KBR (5 micromol L(-1)) in the perfusate induced a slight but not significant decrease in pressure developed and pressure-time integral in steady-state contractions. As it was previously described, the heat involved in a heart muscle contraction can be decomposed into several components (H1, H2, H3 and H4), but only one (H3) was associated with force generation. While H3 decreased with lithium in both types of contractions, H3/PtI ratio remained unaltered, indicating that the economy for pressure maintenance was unaffected. To further investigate the role of Ca entry on force development, a condition in which the contraction is mainly dependent on extracellular calcium was studied. An 'extra' stimulus applied 200 ms after the regular one in a muscle stimulated at 0.16 Hz induces a contraction with this characteristic (Marengo et al., Am J Physiol 1999, 276, H309). Lithium induced a strong decrease in pressure-time integral and H3 associated with this contraction (43 and 45%, respectively) with no change in H3/PtI ratio. Lithium also reduced (53%) an energy component (H2) associated with Ca cycling. The use of KBR showed qualitatively similar results [i.e. a 33% reduction in pressure-time integral associated with the extrasystole (ES) with no changes in H3/PtI ratio and a 30% reduction in the H2 component]. Li and KBR effects appear to be additive and in the presence of 45 mmol L(-1) Li and 5 micromol L(-1) KBR the extrasystole was abolished in 77%. Lithium and KBR effects particularly for the extrasystole can be explained through the inhibition of Ca entry via Na-Ca exchange giving support to the participation of the Na-Ca exchanger in the Ca influx from the extracellular space. In addition, the results also suggest the possibility of an effect of Li on an additional Ca sensitive locus (different than the Na-Ca exchanger). In this connection, in isolated contractions lithium decreased the energy release fraction related to mitochondrial processes (H4) increasing the economy of the overall cardiac contraction.

Heat production under no-flow ischemia (ISCH) and under hypoperfusion (HYP) conditions was measured in single isovolumetric contractions of perfused rat ventricles at 25 degrees C. Resting heat production (Hr) and resting pressure decreased when the perfusion rate was reduced from 6 to 1.5 mL min(-1) or lower flows (HYP) and by ISCH. Maximal developed pressure (P) decreased to 29% and 20% of control by HYP at 0.8 mL min(-1) and ISCH, respectively. The tension-independent heat (TIH) fraction attributed to Ca2+-binding, measured during single contractions, decreased under HYP with an increase in the ratio between the maximum relaxation rate and P (-P/P ratio). The TIH fractions (attributed to Ca2+ binding and Ca2+ removal processes) decreased under ISCH. The long duration TIH fraction associated with Ca2+-dependent mitochondrial activity disappeared at flow rates of 1.5 mL min(-1) or lower. The ratio between the tension-dependent energy release and P was decreased by ISCH but not by HYP, indicating that under ISCH there was an improvement in contractile economy, but this was not modified by HYP. Overall, the results indicate that no-flow and low-flow ischemias are energetically different models. While the contractile failure under HYP seems to be related to a decrease in myofilament Ca2+ sensitivity, under ISCH it appears to be related to decreased cytosolic Ca2+ availability combined with a more noticeable effect on a fraction of energy that has been attributed to mitochondrial activity. Furthermore, mechanical and energetic responses of both models (i.e., ISCH and HYP) found in the present work were not the same as those previously observed in severe hypoxia so that all these models should not be used indistinctly.

Heart basal metabolism has been classically studied as the energy expenditure of those processes unrelated to mechanical activity and often measured by rendering the heart inactive using cardioplegic solutions (usually by increasing extracellular K concentration ([Kle]). In arterially perfused rat heart (at 25 degrees C), raising [K]e from 7 to 25 mM at a constant extracellular Ca concentration ([Ca]e) (0.5 mM), induced an increase in resting heat production (Hr) from 4.1 +/- 0.3 to 5.1 +/- 0.3 mol. wt g-1. Under 25 mM K additional increase in [Ca]e further increased Hr to 6.0 +/- 0.4, 7.0 +/- 0.4 and 8.3 +/- 0.9 mol. wt g-1 for 1, 2 and 4 mM Ca, respectively. While under 7 mM K perfusion Hr was not affected by 4 microM verapamil, under 25 mM K and 2 mM Ca 0.4 microM verapamil induced a decrease in Hr (-1.6 +/- 0.2 mol. wt g-1, n = 5, P < 0.001). Caffeine increased Hr under 0.5 mM Ca and 7 mM K perfusion (+0.32 +/- 0.06 and +1.19 +/- 0.25 mol. wt g-1 for 1 and 5 mM caffeine respectively), but under 25 mM K conditions Hr was not affected by caffeine 2 mM. Severe hypoxia decreased Hr under both 7 and 25 mM K (3.7 +/- 0.5 to 2.7 +/- 0.4 mol. wt g-1 and 7.0 +/- 0.4 to 2.2 +/- 0.5 mol. wt g-1, respectively) suggesting that the increased Hr associated with the verapamil sensitive fraction of heat released is associated to a mitochondrial mechanism. Therefore, the use of high [K]e overestimates basal values by increasing a verapamil sensitive fraction of the energy released. In addition, high [K]e modifies a caffeine sensitive energy component probably due to a depletion of caffeine-dependent Ca stores.

It is widely accepted that the ionic movement across the different membrane systems (i.e. sarcolemma, sarcoplasmic reticulum, mitochondria), plays a major role on heart muscle metabolism. On the other hand, neither the relative role nor the associated energy expenditure of those mechanisms have been definitively established. Biochemical and biophysical measurements of the different ion exchange mechanisms, have provided data leading to the postulation of different models for both resting and active metabolism of the heart muscle. The present work analyzes, from an energetic standpoint, available biochemical and biophysical data from the literature calculating the range of energy expenditure that should be attributable to each mechanism. Sodium, potassium and calcium movements during either resting and/or active state are particularly analyzed and the fractional role of various organelles (sarcolemma, sarcoplasmic reticulum and mitochondria) discussed. From this analysis and the known amount of energy released (or the amount of oxygen consumed) by the muscle it is possible to determine whether there is enough energy for a given model of ionic exchange during the excitation contraction process. In addition to this analysis a comparatively short review of energetic studies performed under pathological conditions is also presented. In particular, the pathological conditions analyzed are those with an energetic compromise such as heart hypertrophy, ischemia and anoxia in which the alteration of ionic transport mechanisms seems to be playing a major role.

The effects of various extracellular K concentrations ([K]e) on energy expenditure and their relationship to ionic exchange mechanisms under quiescent conditions were investigated in the arterially perfused rat heart. The increase in [K]e (from 6 to 12, 24, or 50 mM K) leads to a rapid increase (results are given per gram dry weight) in resting energy expenditure (+5.9 +/- 0.9, +13.6 +/- 1.1, and +30.0 +/- 2.0 mW/g, respectively) followed by a slow decrease toward a new steady rate of heat production but higher (+2.8 +/- 0.7, +6.3 +/- 0.6, and +10.5 +/- 1.1 mW/g) than that observed under control conditions (21.1 +/- 0.7 mW/g). The increase in [K]e from 6 to 50 mM also induced an increase in K influx (calculated from 86Rb uptake and efflux experiments) of approximately 0.25 mumol.g-1.s-1. If this increased K influx is driven by the Na-K pump, an increase in steady resting heat production of approximately 10 mW/g would be expected. This represents 95% of the increase in steady heat production measured for 50 mM K intervention. The simultaneous increase in the cellular Ca flux (+0.1 mumol.g-1.min-1) can only explain (if driven by the sarcolemmal Ca pump) less than 1% of the steady increase in heat production. The work also shows that the initial, transitory increase in resting heat production induced by increasing [K]e is caffeine sensitive and may be at least partially attributable to a transitory enhanced activity of the sarcoplasmic reticulum.

The effects of caffeine (1mM) on energy expenditure and mechanical parameters in rat and toad perfused heart ventricles were examined at various stimulation frequencies. While in rat muscles caffeine significantly depressed developed tension and maximal rates of contraction and relaxation at all frequencies tested, in toad ventricle a slight positive inotropic effect was observed. Even though caffeine did not alter total contraction time in both preparations, in the rat ventricle the last part of relaxation was prolonged. In rat ventricle in the presence of caffeine, the ratios between active heat production per beat and either developed tension or tension time integral increased at all frequencies tested (+303 +/- 47 microJ.mN-1 x g-1 and +1.21 +/- 0.13 mJ.mN-1 x s-1 x g-1 respectively) indicating a decrease in contractile economy. In toad ventricle no changes on these ratios were observed. The fact that only in rat ventricle caffeine decreased muscle economy suggests that caffeine affects a system that is active in rat ventricle but it is not operative in toad ventricle. This gives support to the hypothesis that if in rat ventricle SR-Ca pump (1 ATP hydrolyzed/2 Ca transported) is inhibited by caffeine cytosolic Ca would have to be removed by alternative mechanisms such as Na-Ca exchanger or sarcolemmal Ca pump both with a higher rate of ATP hydrolysis (1 ATP hydrolyzed/Ca transported) with the consequent decrease in muscle economy. Resting heat production was increased by caffeine in both preparations and the magnitude of the increment (+3.0 +/- 0.6 mW.g-1 and +0.75 +/- 0.21 mW.g-1 for rat and toad ventricle respectively) also correlates with the different degree of SR activity in both species.

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.

The effects of caffeine (1 mmol.l-1) on mechanical and energetic parameters in the arterially perfused interventricular rabbit septa were examined at various frequencies of stimulation. Even though 1 mmol-1 caffeine induced a negative inotropic effect only at stimulation rates higher than 0.33 Hz, relaxation was impaired at all frequencies tested. The ratio between maximum rate of relaxation and developed tension (-Tmax/T) was consistently lowered by caffeine, indicating a more marked effect on relaxation over contraction. In addition, while time-to-peak tension was unaffected by caffeine at the dose used, the last part of the relaxation (i.e., of the contractile event) was prolonged at all frequencies in the presence of the drug. Resting heat production (Hr) was increased in the presence of caffeine (1.6 +/- 0.6 mW.g-1). The ratios between active heat production and either developed tension (Ha/T) or tension time integral (Ha/TtI), increased at all frequencies examined (53.3 +/- 8.5 microJ.mN-1.g-1 and 68.2 +/- 9.9 microJ.mN-1.s-1.g-1, respectively), indicating a lowered economy of the contractile process. This is consistent with the lower ATP/Ca ratio reported for the sarcoreticular Ca pump (i.e., one ATP hydrolyzed/2 Ca transported) with respect to the sarcolemmal mechanisms such as Na-Ca exchanger or the sarcolemmal Ca pump, with an ATP/Ca ratio of 1 to 1. Thus, inhibition of the SR-Ca pump by caffeine would induce a higher rate of ATP hydrolysis with the consequent increase in the Ha/T ratio. As a result of the increase in both Ha/T ratio and Hr induced by caffeine, the ratio between total heat production and developed tension (Ht/T) also increased. Therefore, the contractile process appeared to be more efficient in the presence of an active SR, since it is energetically less costly to generate a given level of isometric tension.

A study has been made of changing external sodium concentration [Na]e, over the range 75 to 200 mmol X l-1, on contractile parameters and heat production in isolated, arterially perfused, interventricular rabbit septa.- The observed changes in maximum rate of contraction with [Na]e, either in the presence of a constant external Ca concentration [Ca]e or in the presence of a constant [Na]e2/[Ca]e ratio, paralleled those observed for tension development (T). On the other hand the maximal rate of relaxation (-Tmax) and the ratio -Tmax/T increased. While the ratio between active heat production and developed tension remained unaltered (0.111 +/- 0.003 mJ X mN-1 X g-1 dry weight), resting heat production increased with [Na]e2 with a slope of 95 +/- 18 mW X g-1 X mol-2 X l2. Under resting conditions, a decrease in [Na]e of 50 mmol X l-1 induced a fall in 42K uptake of about 16 nmol X s-1 X g-1 without changes in 42K efflux, suggesting that such an intervention depresses K influx. If the depressed K influx, induced by a decrease in [Na]e of 50 mmol X l-1, is associated with a decrease in Na-K pump activity, a fall in resting heat production of about 0.64 mW X g-1 would be expected. This represent 56% of the calculated change in the resting heat production, 1.14 +/- 0.22 mW X g-1 (mean +/- one confidence interval), suggesting that some process in addition to a depressed Na-K pump activity may be altered by changes in [Na]e.

In the rat left atrium, electrically driven, a superfusion dropping method was developed. The device essentially consisted of a pair of platinum wire electrodes which held one end of the tissue, while the other end was attached to an isometric tension transducer. Either normal or isotopically labeled solutions were poured on the preparation at preset flow rates by means of a minipump. Effluent was collected fractionally in vials for measurement of radioactivity. Several perfusion flows tested led us to the conclusion that higher rates than 2.26 ml/min did not improve contractile performance. Stability of the preparation was found similar to the classical isolated organ-bath method during a 180 min period of observation. Set-up of the superfused atria reproduced paired determinations of 45Ca efflux, together with the contractile parameters. In addition, the frequency of sampling (intervals of 1 min or less) allowed us to detect changes in the washout pattern of exchangeable 45Ca even during early phases of Ca efflux. Therefore, the method reported herein seems to overcome the serious handicap of the scanty tissue employed.

Effects of inorganic phosphate (Pi) on contractile function, ionic exchange, and cellular energetic state were investigated in isolated, arterially perfused, interventricular rabbit septa. The addition of 10 or 20 mM Pi to a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffered perfusate showed 1) a decrease in developed tension due to a decrease in free Ca concentration; 2) an increase in diastolic tension; 3) a decrease in 42K uptake at the same time that 42K efflux was depressed; 4) an increase of 24Na activity; 5) an increase in tissue 47Ca activity; and 6) a decrease in tissue adenosine 5'-triphosphate (ATP) levels. If a 10-mM Pi intervention was applied without changing the free Ca concentration of the perfusate, the mechanical record showed a transient positive inotropic effect without changes in K efflux. These results are consistent with Pi-induced stimulated mitochondrial Ca uptake and Na-K pump inhibition that combine to increase cellular Ca uptake. The increase in cellular Ca and the decrease in cellular ATP would both tend to increase diastolic tension. In the presence of antimycin A, respiration-dependent Ca uptake is inhibited, and mitochondrial Ca uptake is then maintained at the expense of cellular ATP. This process of mitochondrial Ca uptake could account for the deficiency of ATP and the rigor type contracture that occurs with antimycin A. This energy-deficient state is modified if mitochondrial ATP consumption is blocked by oligomycin, and such treatment prevents the ATP-deficient rigor. Under these conditions, although mitochondrial Ca uptake is blocked by the presence of antimycin A and oligomycin, Ca uptake remains augmented secondary to Na-K pump inhibition by Pi.

The effects of arginine vasopressin (VP), 8 mU/ml, on contractility, heat production and glucose metabolism were investigated in isolated, arterially perfused, interventricular rabbit septa. Rest tension linearly increased in the presence of VP with a slope of 0.095 +/- 0.008 g/min. Developed tension (DT), maximal rate of tension development (+dT/dt)max and maximal rate of relaxation (-dT/dt)max showed a biphasic response in the presence of VP. The first phase showed a rapid decline in all three contractile parameters [DT, (+dT/dt)max and (-dT/dt)max] with a half time of 4.4, 3.3 and 4.6 min, respectively. This rapid decline was followed by a recovery period that occurs with an increase in glycolytic flux and on lactic acid production. Along with the effects on contractile parameters, VP increases the ratio heat production over developed tension (40% over control values), indicating that in the presence of VP "muscle efficiency" decreases since it is energetically more costly to generate a given level of isometric tension. This was further confirmed by experiments performed under low Ca conditions (150 microM Ca Cl2) in which, while VP showed no significant changes on contractile parameters, myocardial heat production and heat production over developed tension increased.

The effects of changing external Ca concentration ([Ca]o) on contractile parameters and heat production were investigated in the interventricular rabbit septa and the dog papillary muscle. Double reciprocal plots of tension development as a function of [Ca]o yielded half-maximal activation values of 1.04 +/- 0.17 and 2.8 +/- 0.7 mM Ca for the septum and papillary muscle, respectively. Resting heat rate was similar in both preparations, 1.9 +/- 0.08 mW . g-1 for the septum and 1.7 +/- 0.07 mW . g-1 for the papillary muscle, and it was not altered by changes in [Ca]o. Active heat production (Ha) normalized per unit of force developed (19 +/- 1.3 microJ . mN-1 . g-1) for the septum and the dimensionless ratio Ha/(To . lo), (0.30 +/- 0.02) for the papillary muscle, where To is the isometric tension and lo, the muscle length, remained unaltered with changes in [Ca]o. Total heat production per beat normalized per unit of force developed (Ht/T) for the septum and the ratio Ht/(To . lo) for the papillary muscle decreased hyperbolically with [Ca]o. Therefore, as a result of the unaltered economy of the contractile system and the unchanged resting heat rate, muscle economy improves as [Ca]o approaches physiological levels. Further increase in [Ca]o, over the physiological levels, can only slightly improve muscle economy.

The sodium (Na) fractions of the dog carotid artery were identified by analyzing the effects of different procedures on 24Na washout curves. Although these never yielded more than three exponential terms, four Na fractions were identified. The fast-exchanging component amounted to 73 mmoles Na/kg w.wt., followed the kinetics of diffusion in a flat sheet with a diffusion coefficient of 6.9 X 10(-6) cm2 X s-1, and remained unaltered under the different experimental conditions. The intermediate component exchanged with a half time (t0.5) of 5.3 min and amounted to 18 mmoles Na/kg w.wt. About one third of this (6 mmoles) was constituted by cellular Na and the remaining by non-cellular Na, probably associated with extracellular structures. These two fractions could be discriminated by: (a) decreasing the rate of exchange of the cellular fraction with ouabain or low temperature (17 degrees C); (b) cell damage by freezing and thawing combined with metabolic poisoning; (c) enzymatic digestion of extracellular structures. When procedures (a) and (c) were combined, the intermediate component entirely disappeared. A third residual component amounted to 0.6 mmoles Na/kg w.wt and exchanged with a t0.5 of 70 min. It was unaffected by cellular damage or enzymatic digestion and was masked by the cellular phase in effluxes conducted at 17 degrees C or under ouabain. Its size decreased by microscopic dissection of the dense adventitia, which is probably its source.

The effects of papaverine upon force of contraction, maximal rate of contraction, maximal rate of relaxation and 45Ca efflux were studied in isolated superfused rat left atria electrically driven at 1 Hz. Papaverine (3 X 10(-5) mol/l, increased developed tension (from 5.35 +/- 1.17 mN to 7.18 +/- 1.51 mN) by 1.8 +/- 0.41 mN (+33%, p less than 0.01) and maximal rate of contraction (+T) by 34.5 +/- 13% (p less than 0.05). In all experimental conditions tested, papaverine increased the rate of 45Ca efflux. The amount by which papaverine increased 45Ca efflux was 175 +/- 41 nmoles . g wet wt-1 and 304 +/- 76 nmoles . g wet wt-1, in the presence and in the absence of caffeine, respectively. The plot of changes in 45Ca efflux versus changes in developed tension fitted to a straight line (r = +0.56, n = 17, p less than 0.05), with a slope and intercept of 42 nmoles Ca . mN-1 . g wet wt-1 and -0.47 mN respectively, suggesting an association between the changes induced by papaverine in force development and increased 45Ca efflux.